The Construction And Application Of A High-throughout Method For Screening Threonine-overproducing Strains

Threonine is one of the eight amino acids essential to human and animal livies.It is widely used in agriculture,medicine,food and animal feed industries.With the steady ex-pansion of the market and the decline of profit rate,it is particularly important to improve the strain level as soon as possible.Most of the industrial strains had been reformed by metabolic engineering based on known knowledge,further improvement of these strains by rational design is very difficult.The traditional mutagenesis and screening approach is ap-plicable for such situation but far from efficient.There have been a few reports about using biosensor to construct high-throughput screening methods to obtain high-yielding strains.However,threonine biosensor remains not reported up to now.In this study,we constructed a threonine biosensor and developed a high-throughput method for screening threonine high-yielding strains.Threonine-inducible promoters were selected based on the former analysis of the pro-tein expression patterns of Escherichia coli MG 1655 in response to different concentrations of threonine by using liquid chromatography-tandem mass spectrometry.A Threonine in-ducible fusion promoter cysJHp was created and tested with the expression of lacZ gene linked to the promoter under different conditions.Then,the biosensors were constructed with the EGFP expression and tetracycline resistance as output signals,and tested respec-tively.As a result,the biosensors were proved to be able to distinguish the strains of dif-ferent threonine-producing abilities.The EGFP-based biosensor was integrated to build a high-throughput screening pro-tocol for hyper-producer of threonine.465 strains were selected by fluorescence activated cell sorter from a mutant cell library of threonine-producing Escherichia coli strain ThrH(pTZL1-egfp)and tested for threonine production capability with microtiter plates.44 strains with higher productivities were obtained from these 465 strains,while no better strains were obtained by randomly pickout from the mutant library as the convential meth-od does.Three of the selected strains were tested in a 5 L fermentor,the total threonine amount and yield of ThrH-27 improved by 8.14%and 7.24%,respectively.This study opens a new way to develop efficient high-throughput screening method for strains producing any metabolite,using a sensor containing the metabolite-inducible pro-moter and a signaling gene.