Engineering a Pseudomonas putida chassis with glycine auxotrophy for effective glyphosate assimilation

MASOTTI, F.; KRINK, N.; LUCCI, M.; GARAVAGLIA, B.S.; GOTTIG, N.; NIKEL, P.; JORGELINA OTTADO

Congreso; XVIII Congreso Samige; 2023

Argentina largely uses glyphosate-based herbicides (GBH) to control weeds during agronomic crops cultivation. Glyphosate (GP) is a member of the group of chemical compounds called phosphonates characterized by a chemically stable C-P bond. The excessive use of GP and its widespread presence in the environment make it essential to have tools to detoxify this molecule, if necessary. In our laboratory, we have successfully isolated bacterial strains from Santa Fe province soils where the herbicide has been repeatedly applied. These strains exhibited a remarkable ability to degrade GBH. We found that the Agrobacterium tumefaciens CHLDO strain was able to assimilate GBH by using the C-P lyase pathway, encoded in the phnFGHIJKLO-duf1045-phnMN cluster. The expression of this cluster has proven to be induced when bacteria are grown in minimal medium with GBH as the only phosphorus source.Since glycine serves as a subproduct in the C-P lyase GP-degradation process, our objective was to create a selection strain based on growth. This strain can be utilized for the heterologous expression of various versions of the phn cluster from A. tumefaciens CHLDO. To achieve this, we employed metabolic engineering to develop a Pseudomonas putida chassis derived from EM42, a glycine auxotroph strain known as SLTB7 (P. putida EM42 ΔserA ΔltaE ΔthiO ΔbenABCD, attTn7::RNApolT7). In this strain, GP may be used to produce glycine for bacterial growth. The overexpression of the phn cluster caused a lower growth inhibition due to the presence of GP than in the control strain (SLTB7 transformed with empty plasmid). However, its expression did not allow the bacteria to grow solely in GP as expected. Therefore, we adopted a strategy akin to Aleatory Laboratory Evolution (ALE) experiments, to systematically enhance the efficiency of the C-P lyase degradation pathway, enabling it to effectively break down GP herbicide. However, during this process, we discovered that the SLTB7 strain exhibited a reversal of its glycine auxotrophic phenotype, leading to the loss of selection pressure. Thus, SLTB7 chassis was re-engineered by introducing new mutations in the glyoxylate shunt pathway that allowed, in a final instance, the generation of a strain that can be evolved to use the GP as sole source of carbon. In this sense, a mutant strain in aceA (which codes for an isocitrate lyase), called SLTBA, was generated. Then, to adapt this chassis to growth-based conditions, the enzymes related to glycine catabolism, particularly serine hydroxymethyl transferase, (GlyA-I/GlyA-II) were overexpressed in SLTBA, or this strain was subjected to ALE experiments in sarcosine or glycine as carbon source in the medium.In summary, the results of these experiments allowed optimization of the auxotrophic P. putida chassis to study the mechanism of C-P lyase pathway encoded in the heterologously expressed phn cluster from A. tumefaciens CHLDO.