Genetic Engineering of Industrial Micro-organisms - Casein hydrolysis by the lactic acid bacteria Lactococcus lactis subsp. cremoris and Lactobacillus rhamnosus and Xylose fermentation by the yeast Saccharomyces cerevisiae

Abstract: Micro-organisms are used industrially by humans, to improve production, either qualitatively or quantitatively, and to enhance profit or value. In cheese production, the starter bacteria hydrolyse casein degradation products generated by rennet. Some bitter peptides accumulate during cheese ripening, and their degradation would improve the flavour of the cheese. Plasmid pHP003 from the starter strain Lactococcus lactis subsp. cremoris HP was characterised. It was 13.4 kbp in size and harboured the gene encoding lactocepin, an endopeptidase that hydrolyses casein. The lactocepin gene was linked with a partially deleted abiB gene. Growth and milk acidification rates and lactocepin activities were assessed in strains HP, LW1484 and a HP derivative in which pHP003 had been replaced by the medium-sized (34 kbp) lactocepin plasmid from LW1484. The growth and acidification rates of the HP derivative in milk were higher than for both wild-type strains. However, wild-type HP with pHP003 exhibited significantly higher lactocepin activity than the two strains harbouring the medium-sized lactocepin plasmid. Therefore, the size of the lactocepin plasmid may be important in determining the level of proteolytic activity. Oligopeptidase O from the non-starter strain Lactobacillus rhamnosus HN001 was characterised genetically and biochemically. The pepO gene encodes a protein of 70.6 kDa, which includes the HEXXH motif present in metalloendopeptidases. The purified oligopeptidase O demonstrated a unique cleavage specificity against fragment 1-23 of as1-casein, hydrolysing bonds Pro-5-Ile-6, Lys-7-His-8, His-8-Gln-9, and Gln-9-Gly-10. The novel activity of this enzyme is likely to contribute to the degradation of bitter peptides in cheese production. For fuel-ethanol production from ligno-cellulose hydrolysate, the aim is to achieve as high an ethanol yield as possible. Therefore it is important to enable fermentation of all carbohydrates present in the raw material. Since xylose may constitute up to 23% of the dry weight of the raw material the aim was to develop xylose-fermenting strains. The effect of XKS1 over-expression was determined in two different Saccharomyces cerevisiae host strains, H158 and CEN.PK, also expressing XYL1 and XYL2 from Pichia stipitis. Fermentation was carried out in defined and complex media containing a defined sugar mixture or birch-wood ligno-cellulosic hydrolysate. XKS1 over-expression improved xylose fermentation for both strains in all types of media. However, XKS1 over-expression reduced xylose consumption. Complex medium increased xylose consumption compared with defined medium and increased the ethanol yield. The CEN.PK strain consumed more xylose but accumulated more xylitol than the H158 strain, and thus gave lower ethanol yields from consumed xylose. Therefore, the strain background is also important for generating efficient xylose-fermenting, recombinant Saccharomyces cerevisiae. To quantitatively analyse metabolic fluxes in recombinant Saccharomyces cerevisiae during the metabolism of xylose/glucose mixtures a stable xylose-utilising recombinant strain, TMB 3001, was constructed. XYL1, XYL2, and XKS1 were integrated into the chromosomal his3 locus of Saccharomyces cerevisiae. The strain was stable for more than forty generations in continuous fermentation. Anaerobic ethanol formation from xylose in recombinant Saccharomyces cerevisiae was demonstrated for the first time. However, the strain only grew on xylose in the presence of oxygen.