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Genetic improvement of recombinant protein production in Kluyveromyces lactis. Why Kluyveromyces lactis for biotechnology? It is lactose-positive, able to grow on poor substrates such as whey - It is a safe organism for industrial use (GRAS) characteristics shared by regulated S. cerevisiae Strong and promoters and engineered vectors are available It has good secretory properties It is capable of secreting large proteins into the medium To identify the best genetics and physiological conditions for the production of the heterologous protein we choose as model system the glucoamylase from Arxula adeninivorans Secretion efficiency can be directly detected on starch plates as clear halo by the digestion of starch around the cell Ampr URA3 Sal I P-GAP Hind III Cla I GAA pTS32x-GAA Sma I B (11.69 kb) EcoR I Sal I EcoR I T-PHO5 C Hind III Sma I Bui et al., A Improvement of heterologous protein production Choice of the strain (secretion phenotype) Control of host physiology (carbon source) starch the level of glucoamylase produced and PM4-4B secreted into the medium is JA6 strain dependent 2359 MW270 and carbon source dependent Starch + lactose Starch + glucose starch PM4-4B JA6 2359 MW270 Starch + lactose Starch + glucose JA6 is the best producer starch PM4-4B JA6 2359 MW270 Starch + lactose Starch + glucose Lactose is the best condition and glucose the worst starch PM4-4B JA6 2359 MW270 Starch + lactose Starch + glucose The reduced halo dimension on glucose respect to that observed on starch suggest that glucoamylase productionwas glucose-repressed Glucose 0,2% 1% 5% Effect of glucose concentration the level of heterologous protein produced on gluco-amylase production and secreted into the medium depends on glucose concentration We have therefore analysed the level of secreted glucoamylase in the non repressible mutants dgr151, rag1kht2 and mig1. Gene Coded protein Mutant phenotype RAG1* Coded protein Coded protein DGR151 Coded protein Coded protein MIG1 Coded protein Coded protein * Il locus RAG1 è polimorfico. In JA6 un secondo gene, KHT2, è arrangiato in tandem a RAG1. starch rag1hxt1 dgr151 starch + lactose wild type mig1 starch + glucose rag1hxt1 and dgr151 mutation positively affects production in glucose confirming the glucose repression of production. starch rag1hxt1 dgr151 starch + lactose wild type mig1 starch + glucose dgr151show a better production also in starch and lactose suggesting a role of DGR151 independent of its role in glucose repression starch rag1hxt1 dgr151 starch + lactose wild type mig1 starch + glucose mig1 is severely affected in all the conditions suggesting a positive and consistent role of MIG1 dgr151 is able to iperproduce also other secreted recombinant proteins? To answer to this question we analysed the levels of : - Human serum albumin (HSA) - Interleukin 1b (IL-1b) Protein: Human interleukin-1b Cultural condition: YEPD deprived of phosphate Promoter: PHO5 HIL-1b dgr151 show an increased production of H-interleuchina1b Protein: Human serum albumin Cultural condition: YEPD + ethanol 1% Promoter: ADH4 HSA dgr151 show an increased production of Human serum albumin Cell Wall analysis dgr151 JA6 dgr151 do not show morphological defects Cell Wall functionality Saggio Zymoliasi 28° C Saggio Zimoliasi 37°C 120 JA6 100 100 80 JA6 80 % cellule vitali % cellule vitali 90 dgr 60 40 70 dgr 60 50 40 30 20 20 10 0 0 0 10 20 30 Tempo (min) 40 50 60 0 5 10 15 20 25 30 35 40 Tempo (min) dgr151 do not show functional defects 45 50 55 Analysis of dgr151 mutation •Comparison of wild-type and dgr151 protein G176D WT dgr151 R206A Istituto di Genetica, Università di Parma Barbara Neglia Paola Goffrini Iliana Ferrero Claudia Donnini Dipartimento di Biologia Cellulare e dello Sviluppo Università di Roma "La Sapienza" Francesca Farina Francesca La Starza Claudio Palleschi Acknowledgements MINISTERO DELL’UNIVERSITA’ E DELLA RICERCA SCIENTIFICA - Scientific Research Pogramms of rilevant national interest COFIN 2000 Biotechnology of yeasts: optimisation of the production and secretion of heterologus proteins. Coordinator:Prof. Danilo Porro