Stimulation of acetoin production in metabolically engineered Lactococcus lactis by increasing ATP demand.

Having a sufficient supply of energy, usually in the form of ATP, is essential for all living organisms. In this study, however, we demonstrate that it can be beneficial to reduce ATP availability when the objective is microbial production. By introducing the ATP hydrolyzing F1-ATPase into a Lactococcus lactis strain engineered into producing acetoin, we show that production titer and yield both can be increased. At high F1-ATPase expression level, the acetoin production yield could be increased by 10 %; however, because of the negative effect that the F1-ATPase had on biomass yield and growth, this increase was at the cost of volumetric productivity. By lowering the expression level of the F1-ATPase, both the volumetric productivity and the final yield could be increased by 5 % compared to the reference strain not overexpressing the F1-ATPase, and in batch fermentation, it was possible to convert 176 mM (32 g/L) of glucose into 146.5 mM (12.9 g/L) acetoin with a yield of 83 % of the theoretical maximum. To further demonstrate the potential of the cell factory developed, we complemented it with the lactose plasmid pLP712, which allowed for growth and acetoin production from a dairy waste stream, deproteinized whey. Using this cheap and renewable feedstock, efficient acetoin production with a titer of 157 mM (14 g/L) acetoin was accomplished.

A novel cell factory for efficient production of ethanol from dairy waste.

BACKGROUND: Sustainable and economically feasible ways to produce ethanol or other liquid fuels are becoming increasingly relevant due to the limited supply of fossil fuels and the environmental consequences associated with their consumption. Microbial production of fuel compounds has gained a lot of attention and focus has mostly been on developing bio-processes involving non-food plant biomass feedstocks. The high cost of the enzymes needed to degrade such feedstocks into its constituent sugars as well as problems due to various inhibitors generated in pretreatment are two challenges that have to be addressed if cost-effective processes are to be established. Various industries, especially within the food sector, often have waste streams rich in carbohydrates and/or other nutrients, and these could serve as alternative feedstocks for such bio-processes. The dairy industry is a good example, where large amounts of cheese whey or various processed forms thereof are generated. Because of their nutrient-rich nature, these substrates are particularly well suited as feedstocks for microbial production. RESULTS: We have generated a Lactococcus lactis strain which produces ethanol as its sole fermentation product from the lactose contained in residual whey permeate (RWP), by introducing lactose catabolism into a L. lactis strain CS4435 (MG1363 Δ(3) ldh, Δpta, ΔadhE, pCS4268), where the carbon flow has been directed toward ethanol instead of lactate. To achieve growth and ethanol production on RWP, we added corn steep liquor hydrolysate (CSLH) as the nitrogen source. The outcome was efficient ethanol production with a titer of 41 g/L and a yield of 70 % of the theoretical maximum using a fed-batch strategy. The combination of a low-cost medium from industrial waste streams and an efficient cell factory should make the developed process industrially interesting. CONCLUSIONS: A process for the production of ethanol using L. lactis and a cheap renewable feedstock was developed. The results demonstrate that it is possible to achieve sustainable bioconversion of waste products from the dairy industry (RWP) and corn milling industry (CSLH) to ethanol and the process developed shows great potential for commercial realization.