Physiological responses to folate overproduction in lactobacillys plantarum WCFS1.

<p>Abstract</p> <p>Background</p> <p>Using a functional genomics approach we addressed the impact of folate overproduction on metabolite formation and gene expression in <it>Lactobacillus plantarum </it>WCFS1. We focused specifically on the mechanism that reduces growth rates in folate-overproducing cells.</p> <p>Results</p> <p>Metabolite formation and gene expression were determined in a folate-overproducing- and wild-type strain. Differential metabolomics analysis of intracellular metabolite pools indicated that the pool sizes of 18 metabolites differed significantly between these strains. The gene expression profile was determined for both strains in pH-regulated chemostat culture and batch culture. Apart from the expected overexpression of the 6 genes of the folate gene cluster, no other genes were found to be differentially expressed both in continuous and batch cultures. The discrepancy between the low transcriptome and metabolome response and the 25% growth rate reduction of the folate overproducing strain was further investigated. Folate production per se could be ruled out as a contributing factor, since in the absence of folate production the growth rate of the overproducer was also reduced by 25%. The higher metabolic costs for DNA and RNA biosynthesis in the folate overproducing strain were also ruled out. However, it was demonstrated that folate-specific mRNAs and proteins constitute 8% and 4% of the total mRNA and protein pool, respectively.</p> <p>Conclusion</p> <p>Folate overproduction leads to very little change in metabolite levels or overall transcript profile, while at the same time the growth rate is reduced drastically. This shows that <it>Lactobacillus plantarum </it>WCFS1 is unable to respond to this growth rate reduction, most likely because the growth-related transcripts and proteins are diluted by the enormous amount of gratuitous folate-related transcripts and proteins.</p

Batch wt vs hyper +_- pABA Normal int_Filtered fg-bg2

Transcriptional analysis upon the overexpression of the folate gene cluster on a high copy plasmid when compared to an empty vector in the lactic acid bacterium L. plantarum. The transcriptional response was determined for both strains in the presence and absence of pABA thereby using the hybridisation sceme as described in the data processing section

Batch wt vs hyper +_- pABA Normal int_Filtered fg-bg2

Transcriptional analysis upon the overexpression of the folate gene cluster on a high copy plasmid when compared to an empty vector in the lactic acid bacterium L. plantarum. The transcriptional response was determined for both strains in the presence and absence of pABA thereby using the hybridisation sceme as described in the data processing section

Control vs hyper; 25 mM glucose_Filtered fb-bg

Transcriptional analysis upon the overexpression of the folate gene cluster on a high copy plasmid when compared to an empty vector in the lactic acid bacterium L. plantarum. The transcriptional response was determined for both strains in continuous culture in the presence of glucose thereby using the hybridisation sceme as described in the data processing section

Control vs hyper; 25 mM glucose_Filtered fb-bg

Transcriptional analysis upon the overexpression of the folate gene cluster on a high copy plasmid when compared to an empty vector in the lactic acid bacterium L. plantarum. The transcriptional response was determined for both strains in continuous culture in the presence of glucose thereby using the hybridisation sceme as described in the data processing section

Multilevel optimisation of anaerobic ethyl acetate production in engineered Escherichia coli

Background: Ethyl acetate is a widely used industrial solvent that is currently produced by chemical conversions from fossil resources. Several yeast species are able to convert sugars to ethyl acetate under aerobic conditions. However, performing ethyl acetate synthesis anaerobically may result in enhanced production efficiency, making the process economically more viable. Results: We engineered an E. coli strain that is able to convert glucose to ethyl acetate as the main fermentation product under anaerobic conditions. The key enzyme of the pathway is an alcohol acetyltransferase (AAT) that catalyses the formation of ethyl acetate from acetyl-CoA and ethanol. To select a suitable AAT, the ethyl acetate-forming capacities of Atf1 from Saccharomyces cerevisiae, Eat1 from Kluyveromyces marxianus and Eat1 from Wickerhamomyces anomalus were compared. Heterologous expression of the AAT-encoding genes under control of the inducible LacI/T7 and XylS/Pm promoters allowed optimisation of their expression levels. Conclusion: Engineering efforts on protein and fermentation level resulted in an E. coli strain that anaerobically produced 42.8 mM (3.8 g/L) ethyl acetate from glucose with an unprecedented efficiency, i.e. 0.48 C-mol/C-mol or 72% of the maximum pathway yield.</p

Control vs hyper; 25 mM glucose_Filtered fb-bg

Transcriptional analysis upon the overexpression of the folate gene cluster on a high copy plasmid when compared to an empty vector in the lactic acid bacterium L. plantarum. The transcriptional response was determined for both strains in continuous culture in the presence of glucose thereby using the hybridisation sceme as described in the data processing section

Ethyl acetate production by the elusive alcohol acetyltransferase from yeast

Ethyl acetate is an industrially relevant ester that is currently produced exclusively through unsustainable processes. Many yeasts are able to produce ethyl acetate, but the main responsible enzyme has remained elusive, hampering the engineering of novel production strains. Here we describe the discovery of a new enzyme (Eat1) from the yeast Wickerhamomyces anomalus that resulted in high ethyl acetate production when expressed in Saccharomyces cerevisiae and Escherichia coli. Purified Eat1 showed alcohol acetyltransferase activity with ethanol and acetyl-CoA. Homologs of eat1 are responsible for most ethyl acetate synthesis in known ethyl acetate-producing yeasts, including S. cerevisiae, and are only distantly related to known alcohol acetyltransferases. Eat1 is therefore proposed to compose a novel alcohol acetyltransferase family within the α/β hydrolase superfamily. The discovery of this novel enzyme family is a crucial step towards the development of biobased ethyl acetate production and will also help in selecting improved S. cerevisiae brewing strains

Microbial production of short and medium chain esters: Enzymes, pathways, and applications

Sustainable production of bulk chemicals is one of the major challenges in the chemical industry, particularly due to their low market prices. This includes short and medium chain esters, which are used in a wide range of applications, for example fragrance compounds, solvents, lubricants or biofuels. However, these esters are produced mainly through unsustainable, energy intensive processes. Microbial conversion of biomass-derived sugars into esters may provide a sustainable alternative. This review provides a broad overview of natural ester production by microorganisms. The underlying ester-forming enzymatic mechanisms are discussed and compared, with particular focus on alcohol acyltransferases (AATs). This large and versatile group of enzymes condense an alcohol and an acyl-CoA to form esters. Natural production of esters typically cannot compete with existing petrochemical processes. Much effort has therefore been invested in improving in vivo ester production through metabolic engineering. Identification of suitable AATs and efficient alcohol and acyl-CoA supply are critical to the success of such strategies and are reviewed in detail. The review also focusses on the physical properties of short and medium chain esters, which may simplify downstream processing, while limiting the effects of product toxicity. Furthermore, the esters could serve as intermediates for the synthesis of other compounds, such as alcohols, acids or diols. Finally, the perspectives and major challenges of microorganism-derived ester synthesis are presented.</p