Development of a bioprocess for the production of a herbicidal sugar as sustainable alternative to glyphosate

Weeds pose severe threats to agricultural yields and areas used for settlement and transport. Until now, glyphosate is widely applied to reduce undesired plants worldwide [1]. Due to its negative impact on the environment, insects and humans [2-5] and likely prohibition in the EU until the end of 2023 [6], an effective as well as ecologically acceptable alternative is urgently needed. Repeated application of commercially available nontriazine herbicides caused resistant biotypes of weeds, which requires evolved agents for their control [7]. Promising alternatives to replace these harmful or inefficient products are novel herbicidal substances that target pathways in plants, that cannot be bypassed and arrest growth or cause cell death without harmful side effects on non-target organisms [8]. 7-deoxy-sedoheptulose (7dSh) is such a novel herbicidal candidate. This sugar was isolated from culture supernatants of Synechococcus elongatus and has recently been reported to act as an inhibitor of a key enzyme of the shikimate pathway [8] analogous to glyphosate [1]. Yet the cyanobacterium S. elongatus produces very low amounts of 7dSh merely in the stationary phase of cultivation [8] and is therefore not a suitable candidate for an industrial production. Besides, chemoenzymatic synthesis of 7dSh has a low yield (20%) [8] and is not economically feasible due to high substrate costs such as 5-deoxy-D-ribose. Therefore, we aim to develop a microbial process with Streptomyces setonensis as a natural producer strain [8] for the large-scale production of 7dSh as an alternative herbicide. To evolve a scalable, well-characterized bioprocess for an unknown microbial system, firstly, parameters as media composition, phosphate and nitrogen limitation were examined using a high-throughput microbioreactor system (BioLector) which allows online-monitoring of growth. Moreover, a design of experiments (DOE) approach in shake flasks was conducted to investigate the influence of cultivation factors on each other. To verify the influence of oxygen availability and gain a deeper understanding of the metabolism the oxygen transfer rate is measured with RAMOS® (Respiration Activity MOnitoring System). Results from the DOE approach suggest that elevated osmolarity through NaCl addition enables higher product titers whereas nitrogen starvation or addition has no influence. For other Streptomyces strains it is reported that the synthesis of antibiotics and other secondary metabolites is coupled to phosphate limitation [9]. Surprisingly, in contrast, 7dSh synthesis is enhanced by adding 150% more of both phosphate components than in the basic minimal medium [10] and decreased if phosphate starvation occurs. Therefore, the identification of the trigger for the 7dSh production is still ongoing, although the product titer was already increased by factor 100 from mg/L [8] to g/L. The identified beneficial culture conditions lay the foundation for the process scale-up to a stirred-tank reactor and to further improve product concentration by applying process control and defining the optimal operation regime. REFERENCES [1] Duke, S. O. and Powles, S. B. Glyphosate: a once-in-a-century herbicide. Pest. Manag. Sci. 64, 319–325 (2008). [2] Kiefer, J.S.T., Batsukh, S., Bauer, E. et al. Inhibition of a nutritional endosymbiont by glyphosate abolishes mutualistic benefit on cuticle synthesis in Oryzaephilus surinamensis. Commun Biol 4, 554 (2021). [3] Smith, D.F.Q., Camacho, E., Thakur, R. et al. Glyphosate inhibits melanization and increases susceptibility to infection in insects. PLOS Biology 19(5): e3001182 (2021). [4] Farina, W.M., Balbuena, M.S., Herbert, L.T. et al. Effects of the Herbicide Glyphosate on Honey Bee Sensory and Cognitive Abilities: Individual Impairments with Implications for the Hive. Insects 10, 354 (2019). [5] Meftaul, I.M., Venkateswarlu, K., Dharmarajan, R. et al. Controversies over human health and ecological impacts of glyphosate: Is it to be banned in modern agriculture? Environ Pollut. 263(Pt A):114372 (2020). [6] https://www.bmuv.de/themen/wasser-ressourcen-abfall/boden-und-altlasten/bodenschutz-und-altlasten- worum-geht-es/faq-plan-zum-glyphosat-ausstieg [7] LeBaron, H.M., Hill, E.R. Weeds resistant to nontriazine classes of herbicides. In: LeBaron HM, McFarland JE, Burnside OC (eds) The Triazine Herbicides. Elsevier, Chapter 11 (2008) [8] Brilisauer, K., Rapp, J., Rath, P. et al. Cyanobacterial antimetabolite 7-deoxy-sedoheptulose blocks the shikimate pathway to inhibit the growth of prototrophic organisms. Nat Commun 10, 545 (2019) [9] Martin, J.F. Phosphate control of the biosynthesis of antibiotics and other secondary metabolites is mediated by the PhoR-PhoP system: an unfinished story. J. Bacteriol. 186 (16), 5197–5201 (2004) [10] Koepff, J., Sachs, C.C., Wiechert, W. et al. Germination and Growth Analysis of Streptomyces lividans at the Single-Cell Level Under Varying Medium Compositions. Front. Microbiol. 9:2680 (2018)