Identification Of Acetohydroxyacid Synthase(AHAs)in Haloferax Mediterranei

Acetohydroxyacid synthase（AHAs）is a thiamine diphosphate-dependent enzyme that catalyzes branched-chain amino acid synthesis such as valine（Val）,leucine（Leu）and isoleucine（Ile）.It is the key enzyme of the synthetic pathways of branched-chain amino acid using keto acids as substrates.It can condense two molecules of pyruvate to produce acetolactate and eventually synthesize valine and leucine,as well as catalyze the synthesis of isoleucine by condensing one molecule of pyruvate and one molecule of 2-ketobutyric acid.Acetohydroxyacid synthase has been widely studied in bacteria and fungi,but it has not been reported in extremely halophilic archaea.In this paper,Haloferax mediterranei was used as a model strain to identify this enzyme and study its influence on the accumulation of polyhydroxyalkanoates（PHAs）.First,our bioinformatic analysis showed three candidate genes encoding AHAs in H.mediterranei.HFX₁571（ilvN）presumably encodes the small subunit of AHAs;whereas HFX₁572（ilvB）and HFX₆032（ilvB3）encode the large subunits.Using the pyrF efficient gene knockout system established in our laboratory,the related mutants of the above three candidate genes were constructed,including DF50ΔepsΔilvN,DF50ΔepsΔilvB,DF50ΔepsΔilvB3 and DF50ΔepsΔilvNΔilvB.Then,the mutants were subjected to fermentation to explore the effects of the above genes on the branched amino acid synthesis in haloarchaea and its influences on PHAs accumulation.In the fermentation medium without amino acids addition,the growth of mutants DF50ΔepsΔilvN,DF50ΔepsΔilvB and DF50ΔepsΔilvNΔilvB were significantly inhibited compared with the control strain DF50Δeps,while the growth rate of DF50ΔepsΔilvB3 was comparable to the control strain.The results indicated that the acetohydroxyacid synthase was encoded by two adjacent genes HFX₁571 and HFX₁572 and HFX₆032 was a AHAs redundant gene in H.mediterranei.The growth inhibition phenomenon of DF50ΔepsΔilvN,DF50ΔepsΔilvB and DF50ΔepsΔilvNΔilvB were significantly alleviated when branched amino acids（leucine,valine,isoleucine）with different concentrations were supplied in themedium.For example,the growth of DF50ΔepsΔilvNΔilvB was similar to that of control strain DF50Δeps when leucine,valine and isoleucine were added at a concentration of 0.1 g/L,respectively.This results further demonstrated that HFX₁571 and HFX₁572 encode a functional acetohydroxyacid synthase of Haloarchaea.And,the knockout of HFX-1571 or 1572 inhibit primary metabolism of the cells,and thus seriously reduced the accumulation of PHAs.In this paper,we identified a key enzyme,acetohydroxyacid synthase,responsible for branched-chain amino acid synthesis in H.mediterranei.This enzyme consisted of a small subunit IlvN and a large subunit IlvB,which was encoded by HFX₁571 and HFX₁572,respectively.