D-Lactate (LA)-based oligomers (D-LAOs) are unusual oligoesters consisting of D-LA and D-3-hydroxybutyrate that are produced and secreted by engineered Escherichia coli grown on glucose. The cells heterologously express LA-polymerizing polyhydroxyalkanoate synthase and monomer-supplying enzymes. In this study, we attempted to identify the D-LAO secretion route in E. con, which is thought to be mediated by intrinsic membrane proteins. To this end, a loss-of-function screening of D-LAO secretion was carried out using 209 single-gene membrane protein deletants, which are involved in the transport of organic compounds. Among the deletants of the outer membrane-associated proteins, Delta ompF and Delta ompG exhibited diminished D-LAOs secretion and elevated intracellular D-LAO accumulation. When the ompF and ompG expression levels were down- and up-regulated with plasmids harboring these genes, the secreted amounts of the D-LAOs were changed in correspondence with their expression levels. These results suggest that porins mediate D-LAOs transport through the outer membrane. In particular, OmpF is likely to be the major porin involved in the spontaneous secretion of D-LAOS due to the high basal expression of ompF in the parental strain. Among the deletants of the inner membrane-associated proteins, the Delta mngA, Delta argT, Delta macA, Delta citA and Delta cpxA strains were selected by the screening. These genes are also candidate transporters related to D-LAO secretion, suggesting the presence of multiple secretion routes across the inner membrane. To the best of our knowledge, this is the first report on the mechanism of the microbial secretion of oligoesters. (C) 2017, The Society for Biotechnology, Japan. All rights reserved.