Construction Of High Yield Glutathione Engineering Bacteria And Study On Its Metabolites

Glutathione (GSH) is a tripeptide composed of the amino acid residues L-cysteine,L-glutamic acid and glycine. It is one of the main nonprotein antioxidants presenting in livingorganisms and is predominantly found in eukaryotic cells. It plays a vital role in manyphysiological functions and has a number of diverse applications in food, pharmaceutical, andcosmic industries. GSH could be industrially produced by fermentation or enzymatic synthesis.However, an industrial production for GSH has not been set up in China so far, and the supply ofGSH depends on imports from other countries. Therefore, it is very important to achieve theindustrial production of GSH in China.At present, GSH is mainly produced by microbial fermentation with yeast. In mosteukaryotic and Gram-negative bacteria, GSH is biosynthesized by two consecutiveATP-dependent enzymatic reactions. The first and rate-limiting step is the formation ofγ-glutamylcysteine (γ-GC) from L-glutamate and L-cysteine by γ-glutamate cysteine ligase(γ-GCS, GSHI). The second step is catalyzed by glutathione synthetase (GS, GSHII), whichligates glycine to γ-GC, thus forming GSH. Recently, the novel bifunctional enzymes, termedγ-glutamylcysteine-glutathione synthetase (γ-GCS-GS, GshF) have been identified in someGram-positive bacteria, which could catalyze both γ-GCS and GS reactions and were insensitiveto feedback inhibition. The purpose of this study is to construct yeast high-GSH-producingyeasts with a one-step enzyme GshF in host S. cerevisiae W303-1B and to develop a GSHfermentation method. The project comprises five parts:1. The construction of GSH high-producing strains. The GshF gene cloned by SOE-PCRwas used to construct expression vectors with five different promoters (GAP-PC, GAP-SCP,PGK1and GAL1) and transformed in S. cerevisiae W303-1B, respectively. The positivetransformants of w303B/GshF-GAP-SCP, w303B/GshF-GAP-PC, w303B/GshF-PGK1,w303B/GshF-GAL1, were selected by G418selection marker. GSH productions of differentstrains in shake flask cultures were detected using the method of pre-column derivatizationcombined use of reverse phase high-performance liquid chromatography (RP-HPLC). Resultsshowed that the engineering strain w303B/GshF-GAP-SCP presnted high yield of130mg/Lcompared to host with that of60mg/L.2. Optimization of cultural conditions. To improve the fermentation yield of GSH, cultureconditions of flask shaking fermentation were studied. The effects of fermentation time, differentglucose concentrations were carried out, respectively. The optimized basic fermentationparameters of w303B/GshF-GAP-SCP were20g/L of glucose, temperature30℃, shaking speed180rpm, initial OD600value of0.15and3d of incubation time. Under these conditions, yields ofGSH could reach to150mg/L. 3. Effects of amino acid on cell growth and GSH production. Effects of three precursoramino acids added on cell growth and GSH production were investigated in flask shakingfermentation. Results indicated that amino acid significantly influenced GSH production. Basedon this condition, yields of GSH could reach to250mg/L.4. Effect of overexpression of the ABC-type glutathione exporter on the GSH production.The transformants w303B/GshF-GAP-SCP/Adp was constructed, in which Adp gene wasoverexpressed, effect of overexpression of the Adp gene was studied in flask shakingfermentation. Results indicated that overexpression of this gene improved the extracellularglutathione production, and declined the biomass of the engineered strains compared to theplatform host strain.5. Fed-batch fermentation. Fed-batch fermentation was carried out in a10L fermentor,which contained4L YPD liquid YPD medium. Ten percent inoculum (v/v) was transferred intothe bioreactor. Results indicated that w303B/GshF-GAP-SCP9#displayed highly syntheticactivity for glutathione.