| Gram-negative bacterium Escherichia coli has a tripartite cell envelope with a cytoplasmic membrane,a peptidoglycan（PG）layer,and an asymmetric outer membrane containing lipopolysaccharide（LPS）in its outer leaflet.The biogenesis of peptidoglycan and lipopolysaccharide shares the same substrate UDP-Glc NAc.From UDP-Glc NAc,Mur A catalyzes the first reaction for peptidoglycan biosynthesis while LpxA catalyzes the first reaction for lipopolysaccharide biosynthesis.In this study,the genes mur A and lpxA related to peptidoglycan and lipopolysaccharide biosynthesis,and their same cluster genes were overexpressed to constructed a series of mutant strains from Escherichia coli K12 strain MG1655.The metabolic direction of UDP-Glc NAc,the common precursor of both of them,was changed by gene regulation,and the cell growth,morphology,transcription and expression levels of key genes of mutant strains were detected to study the effects of changing the metabolic synthesis of peptidoglycan and lipopolysaccharide on Escherichia coli,and the metabolic regulation strains with excellent characters were screened out as the starting strains for production and fermentation.The main results are as follows:（1）Overexpression of mur A,the first gene for cytosolic synthesis of peptidoglycan,slowed down the growth of E.coli MG1655 and caused morphological defects.Further overexpression of the cytosolic synthetic terminal gene mra Y or mur G could restore the growth and morphological defects.The cell growth of mur A overexpression strains MG1655/p CDF-P1-mur A,MG1655/p CDF-P2-mur A with two promoters of P1 and P2 respectively,and strains MG1655/p CDF-M1L1,MG1655/p CDF-M1L2,MG1655/p CDF-M2L1 and MG1655/p CDF-M2L2 which both overexpressed mur A and lpxA,were obviously affected,and their cell length increased several times and the morphological uniformity were quite poor under the observation of microscope and transmission electron microscope.Strains MG1655/p CDF-mur A/p BAD-mur G and MG1655/p CDF-mur A/p BAD-mra Y that further overexpressed mur G or mra Y on MG1655/p CDF-P2-mur A returned to normal cell growth and morphology.（2）The expression competition exists between the peptidoglycan biosynthesis gene mur A and the lipopolysaccharide biosynthesis gene lpxA,which share the same reaction substrate UDP-Glc NAc.Overexpression of one of this two genes will cause the transcription level and protein expression of the other gene to decrease obviously.RT-PCR and SDS-PAGE were used to detect mur A and lpxA and their corresponding proteins Mur A and LpxA of strains MG1655/p CDF-P1-mur A,MG1655/p CDF-P2-mur A,MG1655/p CDF-P1-lpxA and MG1655/p CDF-P2-lpxA.The results showed that the overexpression of mur A/lpxA would lead to the decline of lpxA/mur A transcription and protein expression.（3）Over-expression of lipopolysaccharide lipid A biosynthesis genes lpxA,lpxD or lpxH could advance cell growth and L-threonine production of E.coli MG1655.In the detection of growth curves of strains MG1655/p BAD-lpxD,MG1655/p BAD-lpxH and MG1655/p BAD-lpxA,the OD600 in the stable phase was 0.20,0.16 and 0.13 higher than that of the wild-type control MG1655/p BAD33,respectively.Furthermore,overexpression plasmid p FW01-thr A*BC-rht C containing the key genes of L-threonine synthesis and transport were transformed into the above three strains,respectively,to form following L-threonine producing strains:MG1655/p FW01-thr A*BC-rht C/p BAD-lpxA,MG1655/p FW01-thr A*BC-rht C/p BAD-lpxD and MG1655/p FW01-thr A*BC-rht C/p BAD-lpxH,for L-threonine fermentation.After 36hours,compared with the wild-type control MG1655/p FW01-thr A*BC-rht C/p BAD,their L-threonine production increased by 17.4%,22.8%and 46.1%respectively.The L-threonine yield of strain MG1655/p FW01-thr A*BC-rht C/p BAD-lpxH was the highest,reaching 3.20 g×L-1. |
