Abstract
Backgroundl-Threonine is an important amino acid for animal feed. Though the industrial fermentation technology of threonine achieved a very high level, there is still significant room to further improve the industrial strains. The biosensor-based high-throughput screening (HTS) technology has demonstrated its powerful applications. Unfortunately, for most of valuable fine chemicals such as threonine, a HTS system has not been established mainly due to the absence of a suitable biosensor. In this study, we developed a HTS method to gain high-yielding threonine-producing strains.ResultsNovel threonine sensing promoters including cysJp and cysHp were discovered by proteomic analyses of Escherichia coli in response to extracellular threonine challenges. The HTS method was constructed using a device composed of the fused cysJp and cysHp as a promoter and a linked enhanced green fluorescent protein gene as a reporter. More than 400 strains were selected with fluorescence activated cell sorting technology from a library of 20 million mutants and tested within 1 week. Thirty-four mutants have higher productivities than the starting industrial producer. One mutant produced 17.95 % more threonine in a 5-L jar fermenter.ConclusionsThis method should play a functional role for continuous improvement of threonine industry. Additionally, the threonine sensor construction using promoters obtained by proteomics analyses is so convenient that it would be easily extended to develop HTS models for other biochemicals.
Highlights
Threonine is the third bulky amino acid in animal feed industry
Selection of a potential promoter capable of responding to threonine We carried out Isobaric tags for relative and absolute quantitation (iTRAQ)-labelled proteomic analyses of E. coli MG1655 cells treated with 0, 11.9, 29.8, 59.5 g/L threonine added in the cultures, respectively, and 1,632 proteins were detected, representing approximately 40–45 % of the predicted proteins in E. coli
When a cutoff of p value less than 0.05 was applied, 12 proteins were selected (Fig. 1), including proteins of the sulfate metabolism branch of the cysteine biosynthesis pathway encoded by the genes cysD, cysN, cysJ, cysI, cysH, components of sulfate transporters CysP and Sbp, ilvC-encoding acetohydroxy acid isomeroreductase in l-isoleucine biosynthesis pathway from threonine, and additional proteins related with stress response such as those encoded by genes sodB, dps, pal and fliY
Summary
Selection of a potential promoter capable of responding to threonine We carried out iTRAQ-labelled proteomic analyses of E. coli MG1655 cells treated with 0, 11.9, 29.8, 59.5 g/L threonine added in the cultures, respectively, and 1,632 proteins were detected, representing approximately 40–45 % of the predicted proteins in E. coli. The results gave hints that the cysJHp promoter is able to sense the intracellular threonine concentration and is a good indicator of the threonine production capacity. To establish a biosensor capable of working with FACS, a plasmid of pTZL2 carrying an egfp gene under the control of the cysJH promoter was constructed and used to transform the ThrH and ThrL strains. More than 40 % of FACS-selected cells produced higher amount of threonine than the control strain (Fig. 4A), while that was only 10 % for the randomly-selected cells (Fig. 4B). The top 44 threonine hyper-productive mutants in the two groups (Fig. 4A, B) were further cultured in 96-well plates, and threonine concentrations were analyzed using the more accurate HPLC method (Fig. 4a, b). The production yield increased from about 0.39 to 0.46 (g threonine/g glucose), with relative improvement of 17.95 %
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