(582bw) Application of 13C Metabolic Flux Analysis to Understanding the Physiology of Engineered Geobacillus Thermoglucosidasius

Application of ¹³C metabolic flux analysis to understanding the physiology of engineered Geobacillus thermoglucosidasius

C.E. Ward* and D.J. Leak

*c.ward10@imperial.ac.uk

* Department of Life Sciences, Imperial College London, South Kensington, London SW7 2AZ.

Geobacillus thermoglucosidasius is a facultative anaerobe capable of mixed acid fermentation, with optimal growth conditions between 45 ºC and 70 ºC. It has emerged as a promising platform for next generation biofuel production from lignocellulosic biomass due to its ability to effectively metabolise pentose and hexose sugars and longer chain oligomers as well as the increased efficiency that can be achieved in the industrial process through use of a thermophile.

Metabolic engineering of the fermentation pathways of the organism has resulted in a 4-fold increase in ethanol yield from glucose, this has been achieved by redirecting metabolic flux from the major fermentation products of lactate and formate through to ethanol. The high ethanol yielding strain, named DL66, has disruption of both the lactate dehydrogenase and pyruvate formate lyase genes and upregulation of the pyruvate dehydrogenase complex under anaerobic conditions. A further, intermediary, strain termed DL44 has been developed, which carries just the lactate dehydrogenase knock out.

¹³C metabolic flux analysis (¹³C-MFA) is a powerful technique that aims to determine the flow of metabolites through the metabolic pathways of the cell using a combination of experimental and computational techniques. Biomass cultured with an isotopically labelled substrate is analysed for the carbon-13 content of stable metabolites and specifically for the distribution of the isotope isomers – or isotopomers, which make up the entire metabolite pool. Proteinogenic amino acids provide an abundant and stable source of isotopomer pools and as such are routinely employed in ¹³C-MFA; the isotope distribution found in amino acids is indicative of the metabolic pathways that have led to their synthesis and thus can be used to trace the flow of carbon in metabolism.

Measured rates of external fluxes from the cell, such as substrate uptake and fermentation product efflux, as well as biomass precursor requirements are used to determine the total flow of carbon into and out of the cell. These are combined with the isotopomer distributions and a metabolic network model detailing the carbon metabolism of interest, with all carbon atom transitions between metabolites, in computational analysis which provides the metabolic flux of the cell.

We have sought to apply ¹³C-MFA to our strains of G. thermoglucosidasius in order to a) develop an intricate understanding of the metabolic flux distribution in our wild type strain, DL33, and the two engineered strains DL44 (Dldh) and DL66 (DldhDpfl ↑pdh) and b) to identify targets for rational metabolic engineering that will contribute to the iterative strain development process, with the intention of increasing the yield or efficiency of bioethanol production in the industrial process.

Experimental data has been obtained by carrying out small-scale continuous cultivations of the three strains under fermentative, microaerobic and aerobic conditions, in order to determine the differences that may exist not only between strains but also through comparison of different states of metabolism. Cells were grown on a mixture of 20% U-¹³C glucose, 80 % naturally labelled glucose and an amino acid mixture consisting of threonine, serine and glutamic acid, required by the organism for growth under oxygen limiting conditions.

Using biomass that has been cultivated to isotopic steady state, the relative abundance and distribution of carbon-13 atoms in the proteinogenic amino acids of the cell were measured using GCMS analysis. Extracted amino acid labelling patterns were corrected for naturally occurring isotopes. External fluxes of substrate uptake and efflux of metabolites and fermentation products were measured by HPLC analysis, to enable absolute values of flux to be calculated.

Metabolic network models of G. thermoglucosidasius have been created reflecting the genetic background of the wild type and engineered strains under both oxygen limited and aerobic growth. Metabolic models have been constrained based on literature-derived biomass precursor requirements of the organism and physiological and thermodynamic considerations, in order to create working models of metabolism of G. thermoglucosidasius for ¹³C-MFA. The 13CFLUX2 suite of programs is being employed to calculate metabolic flux using the metabolic network models.

Preliminary analysis of the isotopomer distribution of the amino acids has revealed previously unobserved characteristics of the strains. Strain DL44 (Dldh) grown under microaerobic and fermentative conditions has revealed a labelling pattern which is similar to that seen under less oxygen limited (but not under oxygen-limited) conditions in other strains, indicating potentially altered flux in this strain compared to the wild type and other engineered strain. Additionally, under both aerobic and fermentative metabolism flux is observed from central metabolism to the supplemented amino acids glutamic acid, threonine and serine, indicating that these anabolic pathways are at least partially active, even under oxygen limiting conditions.

Ongoing metabolic flux analysis of these data and measured external fluxes, using the metabolic network models and 13CFLUX2 software show that these observed differences in isotopomer distribution reflect significant differences in metabolic flux between wild type and engineered strains, which will be presented and discussed.