Growth medium selection and its economic impact on plasmid DNA production

Current developments in gene medicine and vaccination studies are utilizing plasmid DNA (pDNA) as the vector. For this reason, there has been an increasing trend towards larger and larger doses of pDNA utilized in human trials: from 100-1000 μg in 2002 to 500-5000 μg in 2005. The increasing demand of pDNA has created the need to revolutionalize current production levels under optimum economy. In this work, different standard media (LB, TB and SOC) for culturing recombinant Escherichia coli DH5α harbouring pUC19 were compared to a medium optimised for pDNA production. Lab scale fermentations using the standard media showed that the highest pDNA volumetric and specific yields were for TB (11.4 μg/ml and 6.3 μg/mg dry cell mass respectively) and the lowest was for LB (2.8 μg/ml and 3.3 μg/mg dry cell mass respectively). A fourth medium, PDMR, designed by modifying a stoichiometrically-formulated medium with an optimised carbon source concentration and carbon to nitrogen ratio displayed pDNA volumetric and specific yields of 23.8 μg/ml and 11.2 μg/mg dry cell mass respectively. However, it is the economic advantages of the optimised medium that makes it so attractive. Keeping all variables constant except medium and using LB as a base scenario (100 medium cost [MC] units/mg pDNA), the optimised PDMR medium yielded pDNA at a cost of only 27 MC units/mg pDNA. These results show that greater amounts of pDNA can be obtained more economically with minimal extra effort simply by using a medium optimised for pDNA production.

Low carbon fuels and commodity chemicals from waste gases – systematic approach to understand energy metabolism in a model acetogen

Gas fermentation using acetogenic bacteria offers a promising route for the sustainable production of low carbon fuels and commodity chemicals from abundant, inexpensive C1 feedstocks including industrial waste gases, syngas, reformed methane or methanol. Clostridium autoethanogenum is a model gas fermenting acetogen that produces fuel ethanol and 2,3-butanediol, a precursor for nylon and rubber. Acetogens have already been used in large scale industrial fermentations, they are ubiquitous and known to play a prominent role in the global carbon cycle. Still, they are considered to live on the thermodynamic edge of life and potential energy constraints when growing on C1 gases pose a major challange for the commercial production of fuels and chemicals. We have developed a systematic platform to investigate acetogenic energy metabolism, exemplified here by experiments contrasting heterotrophic and autotrophic metabolism. The platform is built from complete omics technologies, augmented with genetic tools and complemented by a manually curated genome-scale mathematical model. Together the tools enable the design and development of new, energy efficient pathways and strains for the production of chemicals and advanced fuels via C1 gas fermentation. As a proof-of-platform, we investigated heterotrophic growth on fructose versus autotrophic growth on gas that demonstrate the role of the Rnf complex and Nfn complex in maintaining growth using the Wood–Ljungdahl pathway. Pyruvate carboxykinase was found to control the rate-limiting step of gluconeogenesis and a new specialized glyceraldehyde-3-phosphate dehydrogenase was identified that potentially enhances anabolic capacity by reducing the amount of ATP consumed by gluconeogenesis. The results have been confirmed by the construction of mutant strains.

Biotechnology in the development of improved phytases

Phytase enzyme supplements are now ubiquitous in the commercial production of a range of livestock, particularly chickens and pigs. Significant effort has been directed over the last two decades towards producing improved enzymes with higher activity, increased stability and at economic levels in industrial fermentations. As such, there are excellent products on the market, but there is a continuing demand for further improvements to drive down costs and for enzyme manufacturers to increase market share. The rapid development of DNA sequencing and gene synthesis technologies has provided ready access to a large number of new and uncharacterised potential phytases. Challenges remain however in identifying and developing those with improved properties.