Development of a biotechnological production system for aromatic building blocks from sugars

Abstract

Biobased economy is becoming a central strategy to decouple industrial production from fossil feedstock and thus to tackle global warming. Consequently there has been an interest in developing technologies for the production of chemicals, materials, and fuels from renewable bio-based feedstock. This can be achieved thanks to industrial biotechnology and metabolic engineering. Protocatechuic acid (PCA) and vanillic acid (VA) are two suitable molecules that can be used as building blocks or precursors (e.g. vanillin) for the production of bio-polymers for durable plastics. In this study, an Escherichia coli BL21(DE3) strain has been engineered to express a putative 3-dehydroshikimate dehydratase (3DHSd) from Pseudomonas putida KT2440, to enable the conversion of 3-dehydroshikimic acid to protocatechuic acid, and a putative catechol-O-methyltransferase (COMT) from Gluconobacter oxydans 621H, to convert protocatechuic acid to vanillic acid. Strains carrying 3DHSd reached a PCA titre of 0.645±0.021 g/L with a maximum yield of 21.77% (mol-PCA/mol-glucose) after 24 hours. However, strains carrying both genes of the newly inserted pathway showed no VA production, suggesting no activity for COMT or different substrate specificity from the predicted one. To increase PCA production, fed-batch cultivation was carried out leading to a PCA titre of 0.949±0.100 g/L after 72 hours and a yield of 7.62% (mol-PCA/mol-glucose). This production system was found to reach higher yields when compared to other studies relying on extensive metabolic engineering and optimized strains. This suggests that growth condition optimization and depletion of intermediates is a more efficient strategy for the production of aromatic molecules when relying on the shikimic acid pathway.

Popular Abstract

Producing plastic from bacteria – a how to guide

The words “plastic” and “pollution” are often put next to each other, but what if this could finally change? The main problems behind plastic are two: its production from oil, a non-renewable resource, and the way we dispose of it, usually by incineration. Switching to renewable resources and improving the molecules that build up plastic to make it more recyclable could be the solution. Over the course of evolution, nature provided several microorganisms with some (microscopic) tools that we could put to use to make this possible. Using bacteria with the right genetic modifications to produce plastic building blocks could be a final answer to these issues.

In order to grow, bacteria only need sugar which is a renewable resource easily available from plants, such as beetroot. To do so we need the right enzymes (the microscopic tools) capable to modify molecules normally synthesized by the bacteria’s metabolism into plastic building blocks. This is the main concept behind a so-called “bio-refinery”. In this study we have first identified two molecules that can be suitable building blocks for plastic and that can be produced using bacteria: protocatechuic acid (PCA) and vanillic acid (VA).

After finding our targets, we had to find two suitable enzymes capable of synthesizing PCA and VA, and this was done after literature and databases research. The first enzyme (3-dehydroshikimate dehydratase, or 3DHSd) capable of producing PCA was found in another microorganism: Pseudomonas putida. A second enzyme capable of converting PCA into VA (catechol-O-methyltransferase, or COMT) was found in Gluconobacter oxydans instead. The DNA information to produce these enzymes carried by these bacteria was then cloned (“copied”) into a circular DNA molecule (plasmid). The plasmid, now carrying the information for the synthesis of the two enzymes, was then inserted in Escherichia coli, another well-known organism. Our bio-refinery was finally ready to produce our molecules.

To test how well our bio-refinery worked we cultivated the bacteria in a solution of salts and glucose (medium). After that, we checked how much of PCA and VA were excreted from the cell into the medium. Unfortunately, only the 3DHSd enzyme worked leading to the production of PCA with a very good efficiency: up to 21% of the glucose metabolized by the bacteria ended up in PCA. To increase the amount of PCA produced, we then switched to another cultivation method, using a bio-reactor. This led to a lower efficiency when converting glucose into PCA but to a higher overall amount in the solution, reaching almost 1 g per litre.

This has proven to be a suitable method for the production of bio-plastic, using renewable resources as a starting material. We will look into optimizing the efficiency of our bio-refinery and try to find an effective method to recover PCA from the medium to overcome PCA toxicity for the bacteria. Finally, we will try to find other candidate enzymes for the production of VA. Hopefully, on the long term, this will lead to a sustainable method for the production of plastic.

Master’s Degree Project Molecular Biology, 60 credits, 2019
Department of Biology, Lund University
Advisor: Rajni Hatti-Kaul
Lund University, Kemicentrum - Bitoechnology department.

• author: Sacchetto, Stefano
• supervisor: Rajni Hatti-Kaul
• organization: Degree Projects in Molecular Biology
• course: MOBN03 20182
• year: 2019
• type: H2 - Master's Degree (Two Years)
• subject: Biology and Life Sciences
• language: English
• id: 8995211
• date added to LUP: 2019-09-17 10:59:27
• date last changed: 2019-09-17 10:59:27