Elucidation of intrinsic biosynthesis yields using 13C-based metabolism analysis

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.

Figure 1

Schematic description of microbial metabolism. Microbes have the ability to co-metabolize diverse feedstock. Dark circles indicate labeled carbon. The enrichment of labeling in the product acts as an indicator for the relative uptake of sugars.

Figure 2

Schematic examples to demonstrate the use of ¹³C-analysis in elucidating the contributions of various carbon substrates towards the final product synthesis. (A) Biosynthesis yield analyzed by feeding cells with ¹³C-substrates (such as fully labeled glucose and acetate). Abbreviations: GAP, Glyceraldehyde-3-phosphate; PYR, pyruvate; KIV, ketoisovalerate. (B) Relative product yields from a primary substrate (a – Isobutanol from glucose in a low performance strain; ab – valine from glucose in a low performance strain; b – Isobutanol from glucose in JCL260; bb – valine from glucose in JCL260) [28]; c – Free fatty acids from acetate in an E. coli strain [30]; d - biomass from glucose in wild type Synechocystis 6803 [32]; e - D-lactate from acetate in engineered Synechocystis 6803 [33]).

Figure 3

Schematic examples illustrate that ¹³C-analysis can be utilized to determine the contributions of various biosynthetic pathways towards final product yield. (A) ¹³C analysis to study the carbon assimilation during syngas fermentation (¹³CO₂, ¹²CO and H₂). Analysis of metabolite labeling patterns can determine CO₂ and CO utilization for pyruvate production. The isotopomer data of pyruvate was used as a demonstration of ¹³C applications for product yield calculations. (B) Threonine and citramalate pathway for the synthesis of 1-butanol. The carbon rearrangement network shows the labeling of 1-butanol from the two biosynthesis pathways, when fed with 1-¹³C pyruvate and ¹³C bicarbonate.

Figure 4

3D illustrations of relationships among theoretical yield, P/O ratio and non-growth associated ATP maintenance. (A) Theoretical Yield as a function of P/O ratio and non-growth associated ATP maintenance without constraining biomass growth (v(8) ≥ 0). (B) Theoretical Yield as a function of P/O ratio and non growth associated ATP maintenance at growth rate v(8) = 3.6. The units of yield and ATP maintenance are ‘g C16:0 fatty acid/g glucose’ and ‘mol ATP /g glucose’ respectively. Under certain circumstances, the energy cannot be balanced for fatty acid or biomass production, resulting zero yield [47].