The work performed and project results are described below for each partner.
COLIPI: Together with COLIPI we aimed to test enforced ATP wasting (EAW) as a strategy to enhance microbial oil production with the oleaginous red yeast Rhodotorula toruloides. A first essential step was the development of a genetic strategy to express the genes of the F1-subunit of the ATPase enzyme (from E. coli) in this organism. The F1-ATPase hydrolyzes ATP to ADP and thus promotes ATP wasting, which is often coupled with increased metabolic activity. Since the partner had not yet established a genetic toolbox for R. toruloides (which was expected to be present at project start), we built a new modular cloning toolbox for R. toruloides to enable chromosomal integration of heterologous genes via homologous recombination (in our case of the F1-ATPase genes (atpAGD) of E. coli). It is similar to our previously developed Golden Gate cloning toolbox for Zymomonas mobilis (Behrendt et al. 2022). However, we were not able to successfully integrate genes in the chromosome of R. toruloides. Close to the end of the project, we were told at the Yeast Lipid Conference 2024 (in Homburg, Germany) that, unlike reported in the literature, the rate for targeted homologous recombination in R. toruloides is too low implying that random chromosomal integration must be used. We could therefore not succeed in generating a R. toruloides strain that expresses the F1-ATPase and testing the effect of EAW was thus so far not possible.
R toruloides accumulates triacylglycerols (neutral lipids) under nutrient limitation (nitrogen, phosphor or sulfur). This behavior favors the use of two-stage processes with a timed nutrient limitation to switch from growth to lipid production and accumulation (a recent perspective article on two-stage processes, also highlighting the role of EAW, was published in Nature Review Bioengineering (Shabestary et al., 2024) with contributions from us). As part of the original project plan, we investigated omics data from chemostat cultivations of R. toruloides with either nitrogen (Zhu et al. 2012) or phosphate limitation (Wang et al. 2018) to generate a library of promoters that can be utilized for coupling heterologous gene expression to nutrient limitations. Such promoters could be useful, for example, to dynamically induce ATP wasting in the lipid production phase after growth.
At the end of the project, we handed over our developed genetic parts to partner COLIPI for further engineering attempts by the company. We also gave an oral presentation at COLIPI and discussed future engineering perspectives for R. toruloides.
BASF: In our first video meeting with collaborators from BASF we presented our genetic strategies to induce ATP wasting in E. coli strains. Isobionics uses purple nonsulfur bacteria (Rhodobacter sphaeroides) in their fermentation processes to synthesize natural aroma ingredients. Given their experience in genetic engineering of these organisms, it was decided that our collaborators would engineer their strains themselves to express the F1-ATPase genes and to induce ATP wasting, based on our previous developments and guidance. Unfortunately, similar as for the R. toruloides, no strain could be constructed so far that would express the ATPase genes.
In parallel, our collaborators from BASF indicated that they would be interested to test EAW for boosting production of itaconic acid in E. coli. Itaconic acid is already produced in the biotech industry via microbial fermentation. It serves as platform chemical, for example as precursor for certain polymers. Our group had previously significantly improved production of itaconic acid in E. coli (Harder et al. 2016 and 2018). We used our developed itaconate production strain (ita36A) and integrated a plasmid in this strain harboring the genes of the F1-ATPase to test the effect of EAW on itaconic acid production. In shake flasks cultivations we could indeed observe a 10 % increase of product yield with EAW. However, it also resulted in a decreased specific (and volumetric) productivity. We varied several cultivation conditions and found that supplementation of bicarbonate to the engineered strain could already rescue the decreased productivity to some degree, while maintaining the increased yield. Further experimental results indicated that mitigating thermodynamic bottlenecks around the tricarboxylic acid cycle together with a more decent expression of the ATPase genes could further improve itaconic acid synthesis in this strain. We plan to continue these investigations beyond the TRANSMETECH project and then to discuss the final results with partner BASF for possible exploitation.
