Controlled pilot development unit-scale fed-batch cultivation of yeast on spruce hydrolysates

Yeast production on hydrolysate is a likely process solution in large-scale ethanol production from lignocellulose. The hydrolysate will be available on site, and the yeast has furthermore been shown to acquire an increased inhibitor tolerance when cultivated on hydrolysate. However, due to over-flow metabolism and inhibition, efficient yeast production on hydrolysate can only be achieved by well-controlled substrate addition. In the present work, a method was developed for controlled addition of hydrolysate to PDU (process development unit)-scale aerobic fed-batch cultivations of Saccharomyces cerevisiae TMB 3000. A feed rate control strategy, which maintains the ethanol concentration at a low constant level, was adapted to process-like conditions. The ethanol concentration was obtained from on-line measurements of the ethanol mole fraction in the exhaust gas. A computer model of the system was developed to optimize control performance. Productivities, biomass yields, and byproduct formation were evaluated. The feed rate control worked satisfactorily and maintained the ethanol concentration close to the setpoint during the cultivations. Biomass yields of 0.45 g/g were obtained on added hexoses during cultivation on hydrolysate and of 0.49 g/g during cultivation on a synthetic medium with glucose as the carbon source. Exponential growth was achieved with a specific growth rate of 0.18 h(-1) during cultivation on hydrolysate and 0.22 h(-1) during cultivation on glucose

Comparison of protein quantification and extraction methods suitable for E-coli cultures

Many different extraction and analysis methods exist to determine the protein fraction of microbial cells. For metabolic engineering purposes it is important to have precise and accurate measurements. Therefore six different protein extraction protocols and seven protein quantification methods were tested and compared. Comparison was based on the reliability of the methods and boxplots of the normalized residuals. Some extraction techniques (SDS/chloroform and toluene) should never be used: the measurements are neither precise nor accurate. Bugbuster extraction combined with UV280 quantification gives the best results, followed by the combinations sonication-UV280 and EasyLyse-UV280. However, if one does not want to use the quantification method UV280, one can opt to use Bugbuster, EasyLyse or sonication extraction combined with any quantification method with exception of the EasyLyse-BCA_P and sonication-BCA_P combinations

Construction and model-based analysis of a promoter library for E. coli: an indispensable tool for metabolic engineering

<p>Abstract</p> <p>Background</p> <p>Nowadays, the focus in metabolic engineering research is shifting from massive overexpression and inactivation of genes towards the model-based fine tuning of gene expression. In this context, the construction of a library of synthetic promoters of <it>Escherichia coli </it>as a useful tool for fine tuning gene expression is discussed here.</p> <p>Results</p> <p>A degenerated oligonucleotide sequence that encodes consensus sequences for <it>E. coli </it>promoters separated by spacers of random sequences has been designed and synthesized. This 57 bp long sequence contains 24 conserved, 13 semi-conserved (W, R and D) and 20 random nucleotides. This mixture of DNA fragments was cloned into a promoter probing vector (pVIK165). The ligation mixtures were transformed into competent <it>E. coli </it>MA8 and the resulting clones were screened for GFP activity by measuring the relative fluorescence units; some clones produced high fluorescence intensity, others weak fluorescence intensity. The clones cover a range of promoter activities from 21.79 RFU/OD₆₀₀ml to 7606.83 RFU/OD₆₀₀ml. 57 promoters were sequenced and used for promoter analysis. The present results conclusively show that the postulates, which link promoter strength to anomalies in the -10 box and/or -35 box, and to the length of the spacer, are not generally valid. However, by applying Partial Least Squares regression, a model describing the promoter strength was built and validated.</p> <p>Conclusion</p> <p>For <it>Escherichia coli</it>, the promoter strength can not been linked to anomalies in the -10 box and/or -35 box, and to the length of the spacer. Also a probabilistic approach to relate the promoter sequence to its strength has some drawbacks. However, by applying Partial Least Squares regression, a good correlation was found between promoter sequence and promoter strength. This PLS model can be a useful tool to rationally design a suitable promoter in order to fine tune gene expression.</p

Dynamic Metabolic Flux Analysis Demonstrated on Cultures Where the Limiting Substrate Is Changed from Carbon to Nitrogen and Vice Versa

The main requirement for metabolic flux analysis (MFA) is that the cells are in a pseudo-steady state, that there is no accumulation or depletion of intracellular metabolites. In the past, the applications of MFA were limited to the analysis of continuous cultures. This contribution introduces the concept of dynamic MFA and extends MFA so that it is applicable to transient cultures. Time series of concentration measurements are transformed into flux values. This transformation involves differentiation, which typically increases the noisiness of the data. Therefore, a noise-reducing step is needed. In this work, polynomial smoothing was used. As a test case, dynamic MFA is applied on Escherichia coli cultivations shifting from carbon limitation to nitrogen limitation and vice versa. After switching the limiting substrate from N to C, a lag phase was observed accompanied with an increase in maintenance energy requirement. This lag phase did not occur in the C- to N-limitation case

MFA for overdetermined systems reviewed and compared with RNA expression data to elucidate the difference in shikimate yield between carbon- and phosphate-limited continuous cultures of E. coli W3110.shik1

The present contribution focuses on the mathematical techniques used to solve steady state metabolic models for the case of an overdetermined system. Even when parts of the system are underdetermined it is possible to solve the model partially and obtain statistically meaningful results. This is illustrated with data gathered from a set of E. coli W3110.shik1 phosphate- or carbon-limited continuous cultures. It is shown that the low yield in shikimate for C-limited cultures is not due to a lower flux going to the shikimate pathway but is caused by a high secretion of byproducts. Carbon-limited cultures could be better for shikimate production than carbon-abundant cultures provided the byproduct secretion is reduced. Finally, flux calculations are compared with RNA expression data