Team:TU Darmstadt/Project/Upscaling

Upscaling

Choice of Organisms

Participating in the manufacturing track, scaling up the production of the desired monomers glycolic acid and ε-caprolactone as well as our biopolymers PLGA and PLGC constitutes an essential part of our project. Therefore, we are considering possibilities on how to further increase the product yield.

One of the major opportunities for improvement of monomer production and upscaling processes involve the selection of a suitable organism. This organism should meet certain criteria. It should be able to produce our three monomers with maximal titers for less costs, in less time, with less waste materials [1]. Most well-established model organisms have some shortcomings of sorts. The organism in general comprises various criteria which are necessary to be fulfilled for our purpose, without running into troubles with interfering cellular mechanisms [1]. We have chosen S. cerevisiae and E. coli, since they are well characterized and currently applied in a large variety of biotechnological processes. Both of them bear the possibility to be genetically modified using standard molecular-biological methods, thereby constructing complex synthetic pathways that subsequently result in product formation.

The combination of easy handling, widely understood metabolism, high tolerance to solvents, and high resistance to oxidative stress [2] also makes Pseudomonas putida one of the current host candidates, suitable for our monomer production [3]. The organism is classified as a gammaproteobacterium and closely related to the widely used model organism E. coli.

It was previously shown that glycolic acid can be directly synthesized from glyoxylate in S. cerevisiae (1). To adapt this approach, we aimed on overexpressing the natively encoded glyoxylate reductase (HprA) and the previously used enzyme homolog from Arabidopsis thaliana (GLYR1) in P. putida.

We started testing if P. putida was an even better production host for glycolic acid. Due to limitation of time, we were only able to validate P. putida KT2440 as a glycolic acid production host. We found the strain can neither utilize glycolic acid nor glyoxylate as carbon source and is therefore, suitable for production of our monomer glycolic acid. As experts, such as DI Dr. Hans Marx from the University of Natural Resources and Life Sciences, Vienna, experienced with P. putida also recommended us to work with this organism. In the future, we still see potential for P. putida as production host for glycolic acid in higher yields.


Genetic and Metabolic Engineering

Targeted gene deletions and overexpression of heterologous genes such as AtGLYR1 from A. thaliana were an integral part of our project. For the production of organic acids, the use of acid-tolerant microorganisms, especially fungal species like yeasts, has proven to be beneficial as the pH can decrease intensively during fermentation [4]. For this reason, an alternative for more efficient production of glycolic acid appears to be Kluyveromyces lactis, which achieves a titer of up to 15 g/L when grown on D-xylose and ethanol [5]. Similar considerations regarding the adaption to high concentrations of organic solvents can be applied to ε-caprolactone production.

Fermentation Process and Extraction

Another important step for optimization of the monomer production, especially when using bacteria in fermentation processes, is the precise definition of the pH value, which has a great influence on cell viability. Carboxylic acids are often excreted by the cell and accumulate up to a critical concentration, inhibiting growth and inducing self-destruction [6]. It is for this reason that the type of fermentation process can impact the titer in industrial applications. Since it is known that, for instance, caprolactone production underlies product inhibition [7], continuous fermentation possesses an advantage over batch and fed-batch cultures. Currently, ε-caprolactone is already produced on an industrial scale in its oligomerized form with a yield of up to 20 g/L, by using the lipase CAL-A [7].

In addition to strain and cultivation approaches, product extraction is still one of the most challenging issues. We performed analytic high-performance liquid chromatography (HPLC) in order to isolate the desired monomers glycolic acid and ε-caprolactone. However, for purification on a large scale, techniques like preparative HPLC need to be used. Possible approaches may also involve coupling of in situ extraction with fermentation.

Just as poly-ε-caprolactone (PCL), PLA and its copolymers PLGA and PLGC are currently industrially produced by companies like Evonik Industries. The reaction vessels are designed to be batch reactors that are emptied after the reaction time. If the conversion of the monomers is close to 100%, purification is no longer necessary and the melt of the product can be directly transferred into an extruder for following processing.

Therefore, we are full of confidence that the successful implementation of the underlying biological approaches into industrial production is a promising possibility and will act on a large scale as a more sustainable and environmentally friendly alternative.


References

  1. ↑ 1.0 1.1 Nikel P. I., Martínez-García E., de Lorenzo V. (2014). Biotechnological domestication of pseudomonads using synthetic biology. Nature Reviews Microbiology 12, 368. [1].
  2. ↑ Kim, J. and Park, W. (2014). Oxidative stress response in Pseudomonas putida. Applied Microbiology and Biotechnology 98, 6933-6946. [2].
  3. ↑ Mückschel, B., Simon, O., Klebensberger J. (2012). Ethylene Glycol Metabolism by Pseudomonas putida. Applied and Environmental Microbiology. 78(24):8531-8539. doi:10.1128/AEM.02062-12. [3].
  4. ↑ Klose, M. C. (2000). Fermentation mit In-situ-Extraktion und In-situ-Elektroextraktion. Univ. Diss, Technischen Universität München. [4].
  5. ↑ Koivistoinen, O. M., Kuivanen, J., Barth, D., Turkia, H., Pitkänen, J.-P., Penttilä, M., & Richard, P. (2013). Glycolic acid production in the engineered yeasts Saccharomyces cerevisiae and Kluyveromyces lactis. Microbial Cell Factories, 12, 82. [5].
  6. ↑ Lund, P., Tramonti, A., & De Biase, D. (2014). Coping with low pH: molecular strategies in neutralophilic bacteria. FEMS Microbiology Reviews, 38, 1091–1125. [6].
  7. ↑ 7.0 7.1 Schmidt, S., Scherkus, C., Muschiol, J., Menyes, U., Winkler, T., Hummel, W., Gröger, H., Liese, A., Herz, H., & Bornscheuer, U. T. (2015). An Enzyme Cascade Synthesis of ε‐Caprolactone and its Oligomers. Angew. Chem. Int. Ed., 54, 2784-2787. [7].