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Development of a Sustainable Route to the Important Platform Chemical 3-Hydroxypropanoic Acid Using Synthetic Biology and a Geobacillus Chassis

Final Report Summary - GEO-HPA (Development of a Sustainable Route to the Important Platform Chemical 3-Hydroxypropanoic Acid Using Synthetic Biology and a Geobacillus Chassis)

3-Hydroxypropionic acid (3-HP) is a platform chemical and ranked 3rd by the US Department of Energy in the list of twelve platform chemicals which can be derived from biomass. Most of the work on biological production of 3-HP has been carried out using glycerol as substrate. There are various problems associated with glycerol as a feedstock and a more promising approach is to use sugar. The most sustainable source of the sugars is lignocellulosic biomass (LCB). LCB is the most attractive renewable resource for the economic production of biochemicals and biofuels. Geobacillus sp are thermophilic aerobic or facultative anaerobic bacteria and are capable of growth over a wide range of temperature (40-70 degrees C). They can ferment both hexose and pentose sugars and oligomers present in LCB. The high growth temperatures reduces the risk of contamination by other microorganisms, make the process more viable by reducing cooling costs during fermentation and also confers some desirable properties to the growth medium. The current study was undertaken to design a bioprocess for large scale production of 3-HP from sugars using Geobacillus thermoglucosidasius (GBT) as chassis and then extend it to LCB as feedstocks.

Results obtained in the current study:
1. Growth kinetics of GBT: A growth medium was designed for GBT and GBT was grown on different carbon sources (glucose, xylose & glycerol). Glucose and xylose are two most abundant sugars in LCB while glycerol is major by-product of biodiesel industry. GBT was cultured under four different aeration conditions with each carbon source in shake flask. The cell OD_600 reached more than 20 under highly and moderately aerobic conditions while it did not increase beyond 2.0 under microaerobic and anaerobic conditions. The maximum impact of aeration condition was on glycerol. However, the strain showed very little or almost no growth on growth on glycerol under microaerobic and anaerobic conditions. The highest specific growth (2.6 h^-1) was obtained under highly aerobic condition when glucose was used as a carbon source. The achieved growth rate was comparable to that of E. coli. The main metabolites obtained during fermentation were acetate, lactate and ethanol. Acetate and lactate were the main products under aerobic conditions while ethanol was the major product under microaerobic conditions. The amount of organic acids produced on sugars were significantly higher than on glycerol. The pH profiles were quite similar under all the conditions. The pH dropped to less than 5.5 in case of sugars while cultivation on glycerol does not decrease significantly. Cell coagulation took place under all the aeration conditions when strain was grown on sugars after achieving a certain cell growth.

2. Consumption of 3-HP by GBT?: Some organisms like Psuedomonas denitrificans, Cupriavidus necator etc assimilate 3-HP produced by them which is a disadvantageous feature. In order to test whether 3-HP can be consumed by GBT or not, shake flask experiments were conducted in the presence of different concentrations of 3-HP from time zero. It was found that only glucose was exhausted while 3-HP was not consumed in case of mixture while there was no growth and no change in concentration of 3-HP when only 3-HP was used as a carbon source. These results indicate that GBT cannot metabolize/degrade 3-HP which is an advantageous feature of GBT.

3. Identification of suitable enzymes: The literature review was done to find out the enzymes involved in Malonyl-CoA and β-alanine pathway, which could function efficiently in a thermophilic system.

4. Synthesis of genes: After identification of enzymes, genes encoding these enzymes were synthesized. All the genes were codon optimized with respect to Geobacillus thermoglucosidasius (GBT), His-tagged and in a bio-brick format.

5. Construction of operons: The synthesized genes were used to constructs operons for each pathway. Initial cloning work was done in E. coli using two different vectors. All the operons were initially constructed in pJ201 plasmid. Thereafter, the operons were cloned into pMTL-gSLIM-S vector. Finally, the vector containing operon was extracted from E. coli and transformed into G. thermoglucosidasius. The LDH promoter and same RBS sequence was used in all the operons.

6. Malonyl-CoA pathway: Three genes encoding for acetyl-CoA carboxylase (ACC), malonyl-CoA reductase (MCR) and 3-HP dehydrogenase (3-HPD) were cloned to make drive the 3-HP synthesis via this pathway. Using three genes, seven different operons were constructed. The expression of all the proteins was confirmed through western blotting. To make the pathway functional, two different approaches were used. In the first one the acc gene was integrated into genome at pyrE locus using allele coupled exchange technology while the other two genes were cloned into plasmid. In the second one, bifunctional mcr was integrated into genome and acc was put into the plasmid. The six recombinant strains generated this way were tested for 3-HP production at shake flask level. The 3-HP production shown by these strains were quite similar and concentration varied from 3-5 mM. One PhD student Ms Zeenat Bashir helped me in this work. She is continuing this work, so further work is under progress. We have to finish few more experiments and planning to write one publication from this work.

7. β-Alanine pathway: The pathway was divided into two parts; from pyruvate to β-alanine (part I) and β-alanine to 3-HP (part II). The total eight operons were constructed for part I while six were designed for part II. The western blotting confirmed the expression of all the proteins. Due to limited availability of plasmids for GBT, the plasmid-based cloning of two parts could not be on possible. One PhD student Mr Lau Matthew is working on this aspect. He is developing genetic toll box for GBT. In addition, he is using CRISPR method to delete genes from GBT. After developing the one more plasmid, he will continue the work on β-alanine pathway.

GBT is a promising thermophile capable of metabolizing glucose and xylose, two major sugars in lignocellulosic biomass. The organism shows many valuable properties in context to 3-HP production. The high cell growth and growth rate of GBT is comparable to E. coli. In addition, it does not consume or degrade 3-HP as carbon source like some other organisms. In the current study, recombinant GBT strains harbouring 3-HP synthetic pathway for production of 3-HP from sugars were constructed and as a proof of concept of small titre of 3-HP (3-5 mM) was achieved. More work for metabolic engineering of strain followed by process engineering is required. The results obtained in the current study indicates that GBT is a potential organism for 3-HP production and synthesis of 3-HP can be elevated to a high level. Further work in this direction is under progress.