IN-SILICO ANALYSIS OF TERPENOIDS IN SACCHAROMYCES CEREVISIAE

Heterologous production of terpenoids from plants which are of medicinal and industrial interests is getting much attention. For this purpose saccharomyces cerevisiae is the most commonly used host but the yield of terpenoids is much lower. The main aim of this review is to study the terpenoid pathways of saccharomyces cerevisiae, effect of respective host metabolism as well as to study the effect of different carbon sources in silico by means of elementary mode analysis. The production and yield of IPP was main focus point in order to find out the novel metabolic engineering strategy for increasing production of terpenoids. With glucose acting as a substrate, MVA pathway has low potential to produce terpenoids as compared to DXP pathway if we consider formation of precursor. Moreover the carbon source also has impact on yield with nonfermentable source providing more biomass. At last several knock out methodologies were being employed which identified minimal cuts sets for enhanced growth of terpenoids.

Genome-scale metabolic rewiring to achieve predictable titers rates and yield of a non-native product at scale

Achieving high titer rates and yields (TRY) remains a bottleneck in the production of heterologous products through microbial systems, requiring elaborate engineering and many iterations. Reliable scaling of engineered strains is also rarely addressed in the first designs of the engineered strains. Both high TRY and scale are challenging metrics to achieve due to the inherent trade-off between cellular use of carbon towards growth vs. target metabolite production. We hypothesized that being able to strongly couple product formation with growth may lead to improvements across both metrics. In this study, we use elementary mode analysis to predict metabolic reactions that could be targeted to couple the production of indigoidine, a sustainable pigment, with the growth of the chosen host, Pseudomonas putida KT2440. We then filtered the set of 16 predicted reactions using -omics data. We implemented a total of 14 gene knockdowns using a CRISPRi method optimized for P. putida and show that the resulting engineered P. putida strain could achieve high TRY. The engineered pairing of product formation with carbon use also shifted production from stationary to exponential phase and the high TRY phenotype was maintained across scale. In one design cycle, we constructed an engineered P. putida strain that demonstrates close to 50% maximum theoretical yield (0.33 g indigoidine/g glucose consumed), reaching 25.6 g/L indigoidine and a rate of 0.22g/l/h in exponential phase. These desirable phenotypes were maintained from batch to fed-batch cultivation mode, and from 100ml shake flasks to 250 mL ambr® and 2 L bioreactors.

Principal Metabolic Flux Mode Analysis

MotivationIn the analysis of metabolism using omics data, two distinct and complementary approaches are frequently used: Principal component analysis (PCA) and Stoichiometric flux analysis. PCA is able to capture the main modes of variability in a set of experiments and does not make many prior assumptions about the data, but does not inherently take into account the flux mode structure of metabolism. Stoichiometric flux analysis methods, such as Flux Balance Analysis (FBA) and Elementary Mode Analysis, on the other hand, produce results that are readily interpretable in terms of metabolic flux modes, however, they are not best suited for exploratory analysis on a large set of samples.ResultsWe propose a new methodology for the analysis of metabolism, called Principal Metabolic Flux Mode Analysis (PMFA), which marries the PCA and Stoichiometric flux analysis approaches in an elegant regularized optimization framework. In short, the method incorporates a variance maximization objective form PCA coupled with a Stoichiometric regularizer, which penalizes projections that are far from any flux modes of the network. For interpretability, we also introduce a sparse variant of PMFA that favours flux modes that contain a small number of reactions. Our experiments demonstrate the versatility and capabilities of our methodology.AvailabilityMatlab software for PMFA and SPMFA is available in https://github.com/ aalto-ics-kepaco/[email protected], [email protected], [email protected], [email protected] informationDetailed results are in Supplementary files. Supplementary data are available at https://github.com/aalto-ics-kepaco/PMFA/blob/master/Results.zip.

Principal elementary mode analysis (PEMA)

Principal elementary mode analysis (PEMA), provides an easy way to identify metabolic patterns in large fluxomics datasets in terms of the simplest pathways of the organism metabolism