Harnessing phosphonate antibiotics argolaphos biosynthesis enables a synthetic biology-based green synthesis of glyphosate

Glyphosate, N-(phosphonomethyl)glycine, is a globally dominant herbicide, despite concerns regarding its carcinogenic potential and environmental impact. Current industrial glyphosate synthesis uses toxic reagents and solvents, generates significant harmful waste, and necessitates substantial investments in waste processing. Global glyphosate production exceeds one million tons annually, with demand remaining high despite restrictions in residential use. Stricter environmental regulations further necessitate a greener, more efficient glyphosate production method. Biocatalysis offers an environmentally friendly approach, leveraging biological systems' ability to create complex structures with high chemo-, regio-, and stereoselectivity. Advances in synthetic biology now enable manipulation of microbial metabolic pathways for industrial production of valuable chemicals, as exemplified by artemisinic acid and opiate production. However, glyphosate is not naturally produced, requiring a novel approach.

Previous research identified two phosphonopeptides, argolaphos A and B, from *S. monomycini* NRRL B-24309, containing aminomethylphosphonate (AMP). AMP is known as a shunt metabolite in phosphinothricin-producing strains and a product of glyphosate decomposition. Studies on genetic elements like promoters, ribosome-binding sites (RBSs), and insulators have advanced synthetic biology techniques enabling precise control over gene expression. These tools can address challenges in metabolic pathway engineering, such as interference between regulatory elements. This prior knowledge formed the foundation for developing a bio-based glyphosate synthesis strategy.

This study employed a multi-faceted methodology. First, it determined the absolute configuration of argolaphos A using acid hydrolysis, derivatization with Marfey's reagent, and indium-mediated reduction. Next, genome mining identified a gene cluster (*alp*) in *S. monomycini* responsible for AMP biosynthesis. Heterologous expression of this cluster in *S. lividans* confirmed its role, albeit with low AMP production. Optimization involved testing different promoters to drive *alp* gene expression. To understand the AMP biosynthetic pathway, a series of gene combinations were expressed in *S. lividans*, analyzing metabolic profiles via ³¹P NMR. Bottlenecks were identified by comparing expression levels of individual genes and their impact on intermediate accumulation. To further enhance AMP production, a promoter-insulator-RBS strategy was implemented. This involved creating a combinatorial library of strong promoters and RBSs, coupled with insulators to prevent regulatory element interference. Finally, a chemical synthesis route converted AMP to glyphosate via reductive amination using glyoxylic acid and α-pic-BH₃. Various analytical techniques were utilized, including ¹H NMR, ¹³C NMR, ³¹P NMR, HRMS, LC-MS, real-time quantitative PCR, and UPLC/MS for pathway analysis, compound characterization, and quantification.

The absolute configuration of argolaphos A was determined as L-valine and L-N5-hydroxyarginine. The *alp* gene cluster (16 ORFs) was identified as responsible for AMP biosynthesis. Heterologous expression of *alp* in *S. lividans* with a strong promoter (*gapdh*) yielded small amounts of argolaphos A. Analysis of different gene combinations revealed that *alpK* and *alpL* expression was a bottleneck in AMP production. The promoter-insulator-RBS strategy significantly improved AMP production, reaching 52.6 mg/L (a 500-fold increase). The chemical conversion of AMP to glyphosate through reductive amination achieved a 96% yield. Biochemical characterization of individual enzymes (AlpH, Alpl, AlpJ, AlpG, AlpK, AlpL) confirmed their proposed roles in the AMP biosynthetic pathway. The study revealed intricate internal regulation within the *alp* gene cluster, including feedforward and feedback loops affecting gene expression. The developed method successfully combined microbial production of AMP with efficient chemical conversion, offering a more sustainable glyphosate synthesis.

This study successfully addressed the challenge of developing a green synthesis route for glyphosate. The findings demonstrate the power of synthetic biology in manipulating microbial pathways for the production of valuable chemicals. The significant improvement in AMP yield highlights the effectiveness of the promoter-insulator-RBS strategy in overcoming metabolic bottlenecks. The high yield of glyphosate from the chemical conversion step validates the feasibility of the overall process. The results offer a promising alternative to traditional glyphosate production methods, reducing environmental impact and potentially lowering production costs. This approach opens up possibilities for producing other valuable chemicals through similar bio-based synthetic methods.

This research presents a novel, environmentally friendly route for glyphosate synthesis. The identification of the *alp* gene cluster, the optimization of AMP production via synthetic biology, and the development of an efficient chemical conversion to glyphosate represent significant advancements. Future research could focus on further optimizing the microbial production of AMP, exploring alternative chemical conversion methods, and investigating the potential for scaling up this process for industrial applications. The principles outlined here could be applied to the production of other valuable chemicals.

The study focused primarily on optimizing the production of AMP in *S. lividans*, and further optimization for industrial scale-up remains to be explored. The scalability and economic viability of the entire process, including both the biological and chemical steps, need to be evaluated in detail. While the chemical conversion step achieved high yield, the overall process efficiency is influenced by both biological production and chemical conversion steps.