<jats:title>Abstract</jats:title><jats:sec> <jats:title>Background</jats:title> <jats:p>Betaine, an osmoprotective compatible solute, has been used to improve <jats:sc>l</jats:sc>-threonine production in engineered <jats:italic>Escherichia coli</jats:italic><jats:sc>l</jats:sc>-threonine producer. Betaine supplementation upregulates the expression of <jats:italic>zwf</jats:italic> encoding glucose-6-phosphate dehydrogenase, leading to the increase of NADPH, which is beneficial for <jats:sc>l</jats:sc>-threonine production. In <jats:italic>E. coli</jats:italic>, betaine can be taken through ProP encoded by <jats:italic>proP</jats:italic> or ProVWX encoded by <jats:italic>proVWX.</jats:italic> ProP is a H<jats:sup>+</jats:sup>-osmolyte symporter, whereas ProVWX is an ABC transporter. ProP and ProVWX mediate osmotic stress protection by transporting zwitterionic osmolytes, including glycine betaine. Betaine can also be synthesized in <jats:italic>E. coli</jats:italic> by enzymes encoded by <jats:italic>betABIT</jats:italic>. However, the influence of ProP, ProVWX and <jats:italic>betABIT</jats:italic> on <jats:sc>l</jats:sc>-threonine production in <jats:italic>E. coli</jats:italic> has not been investigated. </jats:sec><jats:sec> <jats:title>Results</jats:title> <jats:p>In this study, the influence of ProP, ProVWX and <jats:italic>betABIT</jats:italic> on <jats:sc>l</jats:sc>-threonine production in <jats:italic>E. coli</jats:italic> has been investigated. Addition of betaine slightly improved the growth of the <jats:sc>l</jats:sc>-threonine producing <jats:italic>E. coli</jats:italic> strain TWF001 as well as the <jats:sc>l</jats:sc>-threonine production. Deletion of <jats:italic>betABIT</jats:italic> retarded the growth of TWF001 and slightly decreased the <jats:sc>l</jats:sc>-threonine production. However, deletion of <jats:italic>proP</jats:italic> or/and <jats:italic>proVWX</jats:italic> significantly increased the <jats:sc>l</jats:sc>-threonine production. When <jats:italic>proP</jats:italic> was deleted, the <jats:sc>l</jats:sc>-threonine production increased 33.3%; when <jats:italic>proVWX</jats:italic> was deleted, the <jats:sc>l</jats:sc>-threonine production increased 40.0%. When both <jats:italic>proP</jats:italic> and <jats:italic>proVWX</jats:italic> were deleted, the resulting strain TSW003 produced 23.5 g/l <jats:sc>l</jats:sc>-threonine after 36 h flask cultivation. The genes <jats:italic>betABIT</jats:italic>, <jats:italic>proC</jats:italic>, <jats:italic>fadR</jats:italic>, <jats:italic>crr</jats:italic> and <jats:italic>ptsG</jats:italic> were individually deleted from TSW003, and it was found that further absence of either <jats:italic>crr</jats:italic> (TWS008) or <jats:italic>ptsG</jats:italic> (TWS009) improved <jats:sc>l</jats:sc>-threonine production. TSW008 produced 24.9 g/l <jats:sc>l</jats:sc>-threonine after 36 h flask cultivation with a yield of 0.62 g/g glucose and a productivity of 0.69 g/l/h. TSW009 produced 26 g/l <jats:sc>l</jats:sc>-threonine after 48 h flask cultivation with a yield of 0.65 g/g glucose and a productivity of 0.54 g/l/h, which is 116% increase compared to the control TWF001. </jats:sec><jats:sec> <jats:title>Conclusions</jats:title> <jats:p>In this study, <jats:sc>l</jats:sc>-threonine-producing <jats:italic>E. coli</jats:italic> strains TSW008 and TSW009 with high <jats:sc>l</jats:sc>-threonine productivity were developed by regulating the intracellular osmotic pressure. This strategy could be used to improve the production of other products in microorganisms. </jats:sec>