Abstract
The chiral compound (R)-3-hydroxybutyrate (3HB) is naturally produced by many wild type organisms as the monomer for polyhydroxybutyrate (PHB). Both compounds are commercially valuable and co-polymeric polyhydroxyalkanoates have been used e.g., in medical applications for skin grafting and as components in pharmaceuticals. In this paper we investigate cultivation strategies for production of 3HB in the previously described E. coli strain AF1000 pJBGT3RX. This strain produces extracellular 3HB by expression of two genes from the PHB pathway of Halomonas boliviensis. H. boliviensis is a newly isolated halophile that forms PHB as a storage compound during carbon excess and simultaneous limitation of another nutrient like nitrogen and phosphorous. We hypothesize that a similar approach can be used to control the flux from acetyl-CoA to 3HB also in E. coli; decreasing the flux to biomass and favoring the pathway to the product. We employed ammonium- or phosphate-limited fed-batch processes for comparison of the productivity at different nutrient limitation or starvation conditions. The feed rate was shown to affect the rate of glucose consumption, respiration, 3HB, and acetic acid production, although the proportions between them were more difficult to affect. The highest 3HB volumetric productivity, 1.5 g L−1 h−1, was seen for phosphate-limitation.
Highlights
The commonly used production method for 3HB is hydrolysis of PHB that can be recovered from various types of wild type PHB producing organisms (Lee et al, 1999a,b; Chen and Wu, 2005)
Repeated Batch Cultivations Common cultivation protocols for 3HB production in E. coli have been characterized by the aim of keeping the concentration of carbon source high by the use of the repeated batch concept
Liu et al intermittently added a combination of glucose, ammonium, and magnesium sulfate to the cultivation (Liu et al, 2007) while Gao et al added glucose, yeast extract, and magnesium sulfate by continuous feeding (Gao et al, 2002)
Summary
The commonly used production method for 3HB is hydrolysis of PHB that can be recovered from various types of wild type PHB producing organisms (Lee et al, 1999a,b; Chen and Wu, 2005). This degradation is likely catalyzed by the native thioesterase II (tesB), Figure 1, which in a previous study has been overexpressed to increase 3HB production in E. coli (Liu et al, 2007) It is well-known that the metabolic flux to PHB in wild type cells is a result of a cellular strategy to promote the accumulation of storage carbon under conditions of starvation of a particular nutrient but under excess of a carbon source (Anderson and Dawes, 1990; Doi, 1990; Kim et al, 1994). Fed-batch technology allows control of the cellular growth rate by continuous addition of a selected growth-limiting substance that should here not be the carbon source, which generally is used in industrial processing The limitation makes it possible to avoid the severe effects on cell metabolism resulting from complete starvation and this might have a favorable effect on productivity. This was followed by fed-batch cultivation with glucose in excess, where either phosphorous or nitrogen was limiting and where productivity was studied at a set of relevant feed rates
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