Optimization of Medium Composition for Production of Recombinant Calf Chymosin from Kluyveromyces lactis in Submerged Fermentation

Response surface methodology was applied to optimize medium components for production of recombinant calf chymosin by Kluyveromyces lactis GG799.The previous data indicated that the most suitable carbon source,nitrogen source,salt and vitamin were glucose,yeast extract,KH2PO4 and Ca D-Pantothenate,respectively.The concentration of four media components were optimized by using central composite design of response surface methodology.The optimum medium composition for recombinant calf chymosin production was found to contain glucose 29.84 g· L-1,yeast extract 19.85 g·L-1,KH2PO4 0.1 g·L-1 and Ca D-Pantothenate 4.49 mg·L-1.The enzyme activity of recombinant calf chymosin was 722 U· mL-1,which was in an excellent agreement with the predicted value（723 U·mL-1）.The production of recombinant calf chymosin from Kluyveromyces lactis GG799 was effectively increased by response surface methodology.

Characterization of putative mannoprotein in Kluyveromyces lactis for lactase production

Lactase is a member of theβ-galactosidase family of enzymes that can hydrolyze lactose into galactose and glucose.However,extracellular lactase production was still restricted to the process of cell lysis.In this study,lactase-producing Kluyveromyces lactis JNXR-2101 was obtained using a rapid and sensitive method based on the fluorescent substrate 4-methylumbelliferyl-β-D-galactopyranoside.The purified enzyme was identified as a neutral lactase with an optimum pH of 9.To facilitate extracellular production of lactase,a putative mannoprotein KLLA0_E01057g of K.lactis was knocked out.It could effectively promote cell wall degradation and lactase production after lyticase treatment,which showed potential on other extracellular enzyme preparation.After optimizing the fermentation conditions,the lactase yield from mannoprotein-deficient K.lactis JNXR-2101ΔE01057g reached 159.62 U/mL in a 5-L fed-batch bioreactor.

Genome and metabolic engineering in non-conventional yeasts:Current advances and applications

Microbial production of chemicals and proteins from biomass-derived andwaste sugar streams is a rapidly growing area of research and development.While the model yeast Saccharomyces cerevisiae is an excellent host for the conversion of glucose to ethanol,production of other chemicals from alternative substrates often requires extensive strain engineering.To avoid complex and intensive engineering of S.cerevisiae,other yeasts are often selected as hosts for bioprocessing based on their natural capacity to produce a desired product:for example,the efficient production and secretion of proteins,lipids,and primary metabolites that have value as commodity chemicals.Even when using yeasts with beneficial native phenotypes,metabolic engineering to increase yield,titer,and production rate is essential.The non-conventional yeasts Kluyveromyces lactis,K.marxianus,Scheffersomyces stipitis,Yarrowia lipolytica,Hansenula polymorpha and Pichia pastoris have been developed as eukaryotic hosts because of their desirable phenotypes,including thermotolerance,assimilation of diverse carbon sources,and high protein secretion.However,advanced metabolic engineering in these yeasts has been limited.This review outlines the challenges of using non-conventional yeasts for strain and pathway engineering,and discusses the developed solutions to these problems and the resulting applications in industrial biotechnology.