Towards sustainable plastics : Microbial production of aromatic and aliphatic building blocks from biobased feedstock and plastic hydrolysate

The current plastic system is unsustainable and needs to change. Plastics are mainly produced from fossil feedstock, contributing to greenhouse gas emissions, and low rate of post-consumer recycling results in pollution on land and in oceans with high ecological impact, and huge loss of valuable material. There is an urgent need to find solutions for shifting the raw material base to renewable resources and for recycling the plastics after use. Today, less than 1% of over 350 million tonnes of plastic produced is biobased. This doctoral dissertation presents research performed on developing alternative production routes using microbial cell factories for certain aromatic and aliphatic polymer building blocks for the plastics, from biobased raw and recycled biodegradable polymer. The building blocks comprise protocatechuic acid (PCA), adipic acid and 4- hydroxybutyrate. Protocatechuic acid was produced from glucose using a phenylalanine-overproducing strain of Escherichia coli engineered with the gene 3-dehydroshikimate dehydratase (DSD) from Pseudomonas putida, which catalyses the transformation of 3-dehydroshikimate to PCA. Fed-batch cultivation of the bacteria in a minimal medium resulted in the production of 4.25 g/L PCA, with a molar yield of 18% with respect to glucose. Continuing the cultivation led to conversion of glucose mainly to acetate, indicating a response to stress caused by PCA accumulation. An alternative expression system made use of a randomized promoter library using pSEVA221 plasmid for PCA production in E. coli. A limited screening of the library led to the selection of three novel synthetic constitutive promoters (1.26, 2.7, 2.47) that increased the PCA yield from glucose by 10-21% compared to the inducible T7-promoter used in the previous study. RT-qPCR analysis showed that the DSD gene, regulated by all the synthetic promoters, had high expression during the exponential phase of the cell growth, albeit the gene expression level dropped 250-fold during the stationary phase. Besides the increased product yield, the synthetic promoters avoided the need for a costly inducer.A green route for the production of the C-6 dicarboxylic acid, adipic acid by microbial oxidation of 1,6-hexanediol using the resting cells of Gluconobacter oxydans, was developed. By optimizing various reaction parameters like the strain, pH, aeration and substrate concentration, 37 g/L adipic acid was produced with 99 % yield (mol/mol). Pure adipic acid was recovered by using downstream steps without the use of any solvent.The same G. oxydans strain was also used to produce 4- hydroxybutyrate from 1,4-butanediol, a monomer in the commercially available biodegradable plastic, poly(butylene adipate terephthalate) (PBAT). Plastic films was completely hydrolysed using a thermostable variant of leaf compost cutinase (LCC) at 70 C within 7 days. The monomers terephthalic acid, adipic acid and 1,4-butanediol were also recovered and spererated with purity of 95%, 90% and 62%, respectively. Upcycling of adipic acid was studied by its polymerization with hexamethylenediamine and 1,4-butanediol, respectively, by the immobilized Candida antarctica lipase B, Novozym435.Together, these projects demonstrate not only a way to produce biobased chemicals ar high yields but also a circular economy mindset to resource management that focuses on renewable feedstocks and the reuse of spent materials