Summary: Sophorolipids are promising biological derived surfactants or detergents which find application in household cleaning, personal care and cosmetics. They are produced by specific yeast species and among those, Starmerella bombicola (former Candida bombicola) is the most widely used and studied one. Despite the commercial interest in sophorolipids, the biosynthetic pathway of these secondary metabolites remained hitherto partially unsolved. In this manuscript we present the sophorolipid gene cluster consisting of five genes directly involved in sophorolipid synthesis: a cytochrome P450 monooxygenase, two glucosyltransferases, an acetyltransferase and a transporter. It was demonstrated that disabling the first step of the pathway – cytochrome P450 monooxygenase mediated terminal or subterminal hydroxylation of a common fatty acid – results in complete abolishment of sophorolipid production. This phenotype could be complemented by supplying the yeast with hydroxylated fatty acids. On the other hand, knocking out the transporter gene yields mutants still able to secrete sophorolipids, though only at levels of 10% as compared with the wild type, suggesting alternative routes for secretion. Finally, it was proved that hampering sophorolipid production does not affect cell growth or cell viability in laboratory conditions, as can be expected for secondary metabolites.

Summary: The demand for microbially produced surface‐active compounds for use in industrial processes and products is increasing. As such, there has been a comparable increase in the number of publications relating to the characterization of novel surface‐active compounds: novel producers of already characterized surface‐active compounds and production processes for the generation of these compounds. Leading researchers in the field have identified that many of these studies utilize techniques are not precise and accurate enough, so some published conclusions might not be justified. Such studies lacking robust experimental evidence generated by validated techniques and standard operating procedures are detrimental to the field of microbially produced surface‐active compound research. In this publication, we have critically reviewed a wide range of techniques utilized in the characterization of surface‐active compounds from microbial sources: identification of surface‐active compound producing microorganisms and functional testing of resultant surface‐active compounds. We have also reviewed the experimental evidence required for process development to take these compounds out of the laboratory and into industrial application. We devised this review as a guide to both researchers and the peer‐reviewed process to improve the stringency of future studies and publications within this field of science.