Lysine Acylation And Its Role In Anabolism Regulation

Lysine acylation is a dynamic and reversible post-translational modification,which affects many biological processes through regulating activities,localizations and also interactions of proteins.Lysine acylation links the acyl-CoA metabolism and cellular signalling and its level is highly related with the types and concentrations of various acyl-CoAs.The accumulation of acyl-CoA results in the increasing levels of corresponding lysine acylations through the enzymatic or non-enzymatic mechanism,which implicates various cellular functions.In addition,acyl-CoAs also serve as important precursors for biosynthesis of various classes of products.In this study,we systematically analyzed the occurrence and dynamic change of acylation,and its feedback regulation mechanism involved in biosynthetic pathways.We also demonstrated a newly identified enzymatic acylation modification system allosterically regulated by amino acids,from the perspective that metabolites affecting acylation system.The research includes four main parts:(1)Qualitative and quantitative analysis of malonylome in S.erythraea:We combined the highly sensitive mass spectrometry and high-affinity anti-malonyllysine pan antibodies enrichment to describe a profiling of whole malonylome in S.erythraea.This is the first report about the rough description of malonylome in prokaryotes.After that,we conducted cellular functions analysis,motif analysis,protein-protein interaction analysis and further mimicked the regulatory role of malonylation on acetyl-CoA synthetase and glutamine synthetase.We also quantitatively compared the malonylation levels of the exponential phase and stationary phase in S.erythraea,exploring its malonylation dynamic changes.In addition,We compared the difference of lysine malonylation level and product yield in S.erythraea and S.coelicolor,proving that lysine malonylation level is dependent on the concentration of malonyl-CoA and paving the way for studing the relationship between lysine acylation and microbial biosynthetic pathways.(2)The feedback regulation of lysine acylation on erythromycin biosynthesis:We carried out quantitative comparison of lysine propionylome between wide type strain and high yield strain.The results showed that obvious change in global propionylation levels as compared with acetylation levels.We further explored the lysine propionylation inhibition effect on the enzymatic activity of malonate-semialdehyde dehydrogenase(mmsA2),proving the feedback regulation of lysine propionylation on that enzyme.In addition,we demonstrated that enzymatic activities of aldehyde dehydrogenase and S-adenosylmethionine synthetase were also inhibited by lysine propionylation inhibition in industrial erythromycin fermentation process with excess propanol addition,leading to the decrease in erythromycin yield.Finally,we relieved the inhibition mechanism in high propanol concentration supplement through overexpression of the deacylase.The results indicated that the connection between protein acylation system and microbial biosynthetic pathways was built,which expanded our understanding of regulation medaited by lysine acylation.(3)Existence of regulation mechanism of lysine acylation in various biosynthetic pathways with acyl-CoA as precursors:We first demonstrated that feedback regulation mechanism mediated by lysine acylation existed in the malonyl-CoA dependent undecylprodigiosin alkaloid biosynthetic pathway.At the same time,we also proved it in the production process of biofuel butanol.In addition,we further explored that similar mechanism controlled the biosynthetic pathway of the the artificial synthetic chassis E.coli cell factory for pinosylvin synthesis,which demonstrated that lysine malonylation inhibited the enzymatic activities of 4-coumarate-CoA ligase and stilbene synthase,decreasing the yield of pinosylvin.Furthermore,we partly removed the malonylation inhibition effect by manipulating the malonylated lysine site with arginine substitution,improving the pinosylvin production.The result indicated that the increasing concentrations of acyl-CoA would also elevate the acylation levels,leading to the decrease of products,in addition to its role as the acyl-CoA precursors.The feedback regulation involved in inherent and non-inherent biosynthetic pathways affected the yield of several kinds of nature products.(4)The protein acetyltransferase allosterically regulated by metabolite:On the basis of sudies about the lysine acylation affecting the metabolic pathways,we explored the regulation on lysine acylation mediated by metabolite.We identified a lysine acetyltransferase MaKat,containing a fusion of GNAT domain with an ACT domain in M.aurantiaca.Arginine and cysteine were proved to be ligands by monitoring the conformational changes in the ACT domain in MaKat by using FRET assay.We further demonstrated that cysteine and arginine stimulated the enzymatic activity of MaKat with regard to acetylation of acetyl-CoA synthetase MaAcs,indicating that MaKat was an amino acid allosterically regulated protein acetyltransferase.These findings showed that protein acetylation level and lysine acetyltransferase activity might be tightly related to concentrations of amino acids in actinomyces.These results enriched the field of the lysine acetyltransferase family in prokaryotes,which also provided the basis for understanding the regulatory mechanism on lysine acylation medaited by metabolites.