Abstract
This paper discusses the use of 13C-based metabolism analysis for the assessment of intrinsic product yields — the actual carbon contribution from a single carbon substrate to the final product via a specific biosynthesis route — in the following four cases. First, undefined nutrients (such as yeast extract) in fermentation may contribute significantly to product synthesis, which can be quantified through an isotopic dilution method. Second, product and biomass synthesis may be dependent on the co-metabolism of multiple-carbon sources. 13C labeling experiments can track the fate of each carbon substrate in the cell metabolism and identify which substrate plays a main role in product synthesis. Third, 13C labeling can validate and quantify the contribution of the engineered pathway (versus the native pathway) to the product synthesis. Fourth, the loss of catabolic energy due to cell maintenance (energy used for functions other than production of new cell components) and low P/O ratio (Phosphate/Oxygen Ratio) significantly reduces product yields. Therefore, 13C-metabolic flux analysis is needed to assess the influence of suboptimal energy metabolism on microbial productivity, and determine how ATP/NAD(P)H are partitioned among various cellular functions. Since product yield is a major determining factor in the commercialization of a microbial cell factory, we foresee that 13C-isotopic labeling experiments, even without performing extensive flux calculations, can play a valuable role in the development and verification of microbial cell factories.
Highlights
Recent advances in metabolic engineering have enabled us to engineer microbial cell factories for the efficient synthesis of diverse products, including bulk chemicals, pharmaceutical drugs and biofuels [1,2]
Due to suboptimal energy metabolism, the actual bacterial biosynthesis is often at least three-fold lower than the amount that would be predicted from reaction stoichiometry [15]
To probe the contribution of nonoxidative glycolytic cycle (NOG) pathway to overall cell metabolism, this study has presented a carbon rearrangement map for 13C-analysis of the NOG pathway function
Summary
Recent advances in metabolic engineering have enabled us to engineer microbial cell factories for the efficient synthesis of diverse products, including bulk chemicals, pharmaceutical drugs and biofuels [1,2]. In a recent study of an engineered E. coli for fatty acid overproduction [47], 13C-MFA showed that the theoretical ATP/NADPH generation (assuming P/O ratio = 3) from glucose catabolism was much higher than ATP/NADPH consumption for biomass growth and fatty acid synthesis. After optimization of biosynthesis pathway via 'pushpull-block' strategies, this engineered strain had a fatty acid yield of only 0.17 g fatty acid/g glucose (Figure 4B) because a substantive fraction of energy yield from glucose catabolism was lost due to the suboptimal energy metabolism Such high cell maintenance and low P/O ratio in the engineered E. coli are likely caused by the various physiological stresses during biofuel overproduction (e.g., changed cell membrane integrity and compositions [51]). A more promising approach would be to improve energetic prosperity or respiration efficiency, thereby allowing the cells to “burn” substrates more efficiently to satisfy the energy requirement [52,53]
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