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Especially in case of extracellular abundance, the proper determination of intracellular metabolite levels poses challenges. Efficient removal of extracellular substrates and products is therefore important not to overestimate the intracellular amounts. In this study we evaluated two different rapid sampling methods, one combined with cold filtration and the other with centrifugation, for their applicability to determine intracellular amounts of metabolites which are present in high concentrations in the extracellular medium. The filtration\u2010based method combines fast sampling and immediate quenching of cellular metabolism in cold methanol, with rapid and effective removal of all compounds present outside the cells by means of direct filtration and subsequent filtration\u2010based washing. In the centrifugation\u2010based method, removal of the extracellular metabolites from the cells was achieved by means of multiple centrifugation and resuspension steps with the cold quenching solution. The cold filtration method was found to be highly superior to the centrifugation method to determine intracellular amounts of metabolites related to penicillin\u2010G biosynthesis and allowed the quantification of compounds of which the extracellular amounts were 3\u20134 orders of magnitude higher than the intracellular amounts. Using this method for the first time allowed to measure the intracellular levels of the side chain precursor phenylacetic acid (PAA) and the product penicillin\u2010G of the penicillin biosynthesis pathway, compounds of which the transport mechanism in <jats:italic>Penicillium chrysogenum<\/jats:italic> is still far from being sufficiently understood. Biotechnol. Bioeng. 2010;107: 105\u2013115. \u00a9 2010 Wiley Periodicals, Inc.<\/jats:p>","DOI":"10.1002\/bit.22786","type":"journal-article","created":{"date-parts":[[2010,5,7]],"date-time":"2010-05-07T17:03:08Z","timestamp":1273251788000},"page":"105-115","source":"Crossref","is-referenced-by-count":62,"title":["Intracellular metabolite determination in the presence of extracellular abundance: Application to the penicillin biosynthesis pathway in <i>Penicillium chrysogenum<\/i>"],"prefix":"10.1002","volume":"107","author":[{"given":"Rutger D.","family":"Douma","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lodewijk P.","family":"de Jonge","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Caspar T.H.","family":"Jonker","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Reza M.","family":"Seifar","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Joseph J.","family":"Heijnen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Walter M.","family":"van Gulik","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2010,6,3]]},"reference":[{"key":"e_1_2_6_2_1","doi-asserted-by":"publisher","DOI":"10.1021\/ac0623888"},{"key":"e_1_2_6_3_1","doi-asserted-by":"publisher","DOI":"10.1021\/ac8022857"},{"key":"e_1_2_6_4_1","doi-asserted-by":"publisher","DOI":"10.1007\/s11306-008-0116-4"},{"key":"e_1_2_6_5_1","doi-asserted-by":"publisher","DOI":"10.1021\/ac900999t"},{"key":"e_1_2_6_6_1","doi-asserted-by":"publisher","DOI":"10.1016\/0003-2697(92)90149-2"},{"key":"e_1_2_6_7_1","doi-asserted-by":"publisher","DOI":"10.1002\/bit.22689"},{"key":"e_1_2_6_8_1","doi-asserted-by":"publisher","DOI":"10.1002\/bit.22689"},{"key":"e_1_2_6_9_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.trac.2004.11.021"},{"key":"e_1_2_6_10_1","doi-asserted-by":"publisher","DOI":"10.7164\/antibiotics.42.1398"},{"key":"e_1_2_6_11_1","doi-asserted-by":"crossref","first-page":"2589","DOI":"10.1128\/aem.61.7.2589-2595.1995","article-title":"Penicillium chrysogenum takes up the penicillin\u2010G precursor phenylacetic acid by passive diffusion","volume":"61","author":"Hillenga DJ","year":"1995","journal-title":"Appl Environ Microbiol"},{"key":"e_1_2_6_12_1","doi-asserted-by":"publisher","DOI":"10.1139\/m86-087"},{"key":"e_1_2_6_13_1","doi-asserted-by":"publisher","DOI":"10.1002\/bit.10048"},{"key":"e_1_2_6_14_1","doi-asserted-by":"publisher","DOI":"10.1021\/bi00273a029"},{"key":"e_1_2_6_15_1","doi-asserted-by":"publisher","DOI":"10.1007\/BF00270775"},{"key":"e_1_2_6_16_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.mib.2005.04.009"},{"key":"e_1_2_6_17_1","doi-asserted-by":"publisher","DOI":"10.1002\/bit.10683"},{"key":"e_1_2_6_18_1","doi-asserted-by":"publisher","DOI":"10.1007\/s10529-006-9218-0"},{"key":"e_1_2_6_19_1","doi-asserted-by":"publisher","DOI":"10.1002\/bit.20842"},{"key":"e_1_2_6_20_1","doi-asserted-by":"publisher","DOI":"10.1002\/bit.260390404"},{"key":"e_1_2_6_21_1","doi-asserted-by":"publisher","DOI":"10.1016\/0167-4889(91)90199-8"},{"key":"e_1_2_6_22_1","doi-asserted-by":"crossref","first-page":"153","DOI":"10.1002\/jctb.5020260125","article-title":"Selection of strains of Penicillium chrysogenum with reduced penicillin yields in continuous cultures","volume":"26","author":"Righelato RC","year":"1976","journal-title":"J Appl Chem Biotechnol"},{"key":"e_1_2_6_23_1","doi-asserted-by":"publisher","DOI":"10.1016\/0167-7012(95)00104-2"},{"key":"e_1_2_6_24_1","doi-asserted-by":"publisher","DOI":"10.1007\/BF01837056"},{"key":"e_1_2_6_25_1","doi-asserted-by":"publisher","DOI":"10.1085\/jgp.80.3.377"},{"key":"e_1_2_6_26_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.chroma.2008.02.008"},{"key":"e_1_2_6_27_1","doi-asserted-by":"publisher","DOI":"10.1006\/abio.1993.1452"},{"key":"e_1_2_6_28_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0003-2670(02)00240-4"},{"key":"e_1_2_6_29_1","unstructured":"van den BergMA BovenbergRAL DriessenAJM KoningsWN SchuursTA NieboerM WesterlakenI.2001.Method for enhancing secretion of beta\u2010lactam transport. 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