Upcycling of plastic waste by an engineered microorganism into a novel chemical building block

Plastics are omnipresent in our everyday lives and are used in the most diverse applications. However, their short and linear life cycle gives rise to increasing concern, as plastic waste results in, for example, greenhouse gas emissions and leaching of microplastics. As the majority is still produced from fossil fuels as well, their continued production and use is far from sustainable. In order to provide a solution for this a bacterial host was genetically engineered for the upcycling of polyethylene terephthalate (PET) waste into the value-added compound 2-pyrone-4,6-dicarboxylic acid (PDC). This is a chemical building block that can, for example, be used to make strong but biodegradable polymers. PET can be depolymerised to its constituting monomers ethylene glycol (EG) and terephthalic acid (TPA). Therefore the goal of this research was to engineer the host so it can convert TPA to PDC, whilst using EG as a carbon and energy source. This in order to make PET recycling more economically attractive and valorise this enormous waste stream

Synthetic protein scaffolds for flavonoid production in Saccharomyces cerevisiae

Specialised plant metabolites, including flavonoids, have valuable bioactive properties for human health. Traditional production methods are time consuming, create a huge amount of waste and are dependent on external factors. Micro-organisms are therefore a very promising alternative, allowing production of these molecules on an industrial scale. Despite the extensive knowledge on Saccharomyces cerevisiae, many bottlenecks still remain in the construction of an industrial relevant strain. Improper balancing of heterologous pathway genes and loss of intermediates due to side reactions by native enzymes decrease the overall yield and could lead to the accumulation of toxic intermediates. Substrate channelling through the formation of an enzyme complex could help overcome these problems. The different enzymes of the heterologous pathway can be individually balanced leading to an optimised pathway flow. By combining the heterologous pathway at a specific site, local substrate availability can be improved and intermediates are more efficiently channelled through the pathway, thereby reducing the risk of side reactions. We are therefore developing various of these synthetic protein scaffolds in order to gain more insight in how these scaffolds help to increase production of flavonoids, thereby searching for guidelines to apply these technique to other interesting metabolites

Challenges in the microbial production of flavonoids

As flavonoids have beneficial health effects on humans, they are gaining increasing interest from pharmaceutical and health industries. However, current production methods, such as plant extraction and chemical synthesis, are inadequate to meet the demand. Therefore, microbial production might offer a promising alternative. During recent years, microbial strains able to produce flavonoids to a certain extent have been developed. However production titers are limited to the mg l−1 range, hampering the industrial exploitation of these strains. The latter will not be achieved by simply introducing the heterologous pathway in the production host and optimizing the fermentation process, but will depend on the interaction of different aspects of metabolic engineering and process engineering to overcome the current limitations. Next to engineering the production strain to optimize the availability of precursors, the pathway itself also requires intensive engineering. Currently utilized strategies result in a wide variety of different production strains, requiring high-throughput screening methods to identify optimal performing strains. As more and more organisms are being characterized, each with their own specific properties which might be beneficial for the heterologous production of flavonoids, the choice of the production host is another important aspect. Finally, the use of co-cultures might offer an alternative in which different parts of the process are performed by different organisms. This review aims to provide an overview of the research that has been done on these separate aspects. The work presented here could be used as a framework for further research

Engineering Comamonas testosteroni for the production of 2-pyrone-4,6-dicarboxylic acid as a promising building block

Abstract Background Plastics are an indispensable part of our daily life. However, mismanagement at their end-of-life results in severe environmental consequences. The microbial conversion of these polymers into new value-added products offers a promising alternative. In this study, we engineered the soil-bacterium Comamonas testosteroni KF-1, a natural degrader of terephthalic acid, for the conversion of the latter to the high-value product 2-pyrone-4,6-dicarboxylic acid. Results In order to convert terephthalic acid to 2-pyrone-4,6-dicarboxylic acid, we deleted the native PDC hydrolase and observed only a limited amount of product formation. To test whether this was the result of an inhibition of terephthalic acid uptake by the carbon source for growth (i.e. glycolic acid), the consumption of both carbon sources was monitored in the wild-type strain. Both carbon sources were consumed at the same time, indicating that catabolite repression was not the case. Next, we investigated if the activity of pathway enzymes remained the same in the wild-type and mutant strain. Here again, no statistical differences could be observed. Finally, we hypothesized that the presence of a pmdK variant in the degradation operon could be responsible for the observed phenotype and created a double deletion mutant strain. This newly created strain accumulated PDC to a larger extent and again consumed both carbon sources. The double deletion strain was then used in a bioreactor experiment, leading to the accumulation of 6.5 g/L of product in 24 h with an overall productivity of 0.27 g/L/h. Conclusions This study shows the production of the chemical building block 2-pyrone-4,6-dicarboxylic acid from terephthalic acid through an engineered C. testosteroni KF-1 strain. It was observed that both a deletion of the native PDC hydrolase as well as a pmdK variant is needed to achieve high conversion yields. A product titer of 6.5 g/L in 24 h with an overall productivity of 0.27 g/L/h was achieved

Metabolic engineering for glycoglycerolipids production in E. coli : tuning phosphatidic acid and UDP-glucose pathways

Glycolipids are target molecules in biotechnology and biomedicine as biosurfactants, biomaterials and bioactive molecules. An engineered E. coli strain for the production of glycoglycerolipids (GGL) used the MG517 glycolipid synthase from M. genitalium for glucosyl transfer from UDPGlc to diacylglycerol acceptor (Mora-Buyé et al., 2012). The intracellular diacylglycerol pool proved to be the limiting factor for GGL production. Here we designed different metabolic engineering strategies to enhance the availability of precursor substrates for the glycolipid synthase by modulating fatty acids, acyl donor and phosphatidic acid biosynthesis. Knockouts of tesA, fadE and fabR genes involved in fatty acids degradation, overexpression of the transcriptional regulator FadR, the acyltransferases PlsB and C, and the pyrophosphatase Cdh for phosphatidic acid biosynthesis, as well as the phosphatase PgpB for conversion to diacylglycerol were explored with the aim of improving GGL titers. Among the different engineered strains, the ΔtesA strain co-expressing MG517 and a fusion PlsCxPgpB protein was the best producer, with a 350% increase of GGL titer compared to the parental strain expressing MG517 alone. Attempts to boost UDPGlc availability by overexpressing the uridyltransferase GalU or knocking out the UDP-sugar diphosphatase encoding gene ushA did not further improve GGL titers. Most of the strains produced GGL containing a variable number of glucosyl units from mono-to tetra-saccharides. Interestingly, the strains co-expressing Cdh showed a shift in the GGL profile towards the diglucosylated lipid (up to 80% of total GGLs) whereas the strains with a fadR knockout presented a higher amount of unsaturated acyl chains. In all cases, GGL production altered the lipidic composition of the E. coli membrane, observing that GGL replace phosphatidylethanolamine to maintain the overall membrane charge balance