In vitro metabolic engineering is an emerging framework for bioproduction systems, in which synthetic metabolic pathways are constructed using a limited number of enzymes. Employment of thermophilic enzymes as catalytic elements in pathways enables the use of simple heat purification of recombinantly expressed enzymes. However, thermophilic enzymes are generally incompatible with thermo-labile substrates and intermediates. In previous work, we showed that lactate production through a non-ATP forming chimeric Embden-Meyerhof (EM) pathway required careful adjustment of the metabolic fluxes by continuous substrate feeding and optimization of enzyme ratios to prevent the accumulation and degradation of thermo-labile intermediates (Ye et al., Microb. Cell Fact., 11, 120, 2012). In the study reported here, we constructed an in vitro non-phosphorylative EntnereDoudoroff (np-ED) pathway. Because of the high thermal stability of the metabolic intermediates in the np-ED pathway, it could prevent degradation of accumulated metabolic intermediates caused by inconstant metabolic fluxes, and batch-mode production of lactate in which the concentrations of the substrate and metabolic intermediates change dynamically could be achieved. By combining the enzymes involved in the np-ED pathway and lactate dehydrogenase, 20.9 mM lactate was produced from 10 mM glucose and 1 mM gluconate in 6 h. (C) 2019, The Society for Biotechnology, Japan. All rights reserved.