Lactic acid (LA) is an important and versatile chemical that can be produced from renewable resources such as biomass. LA is used in the food, pharmaceutical, and polymers industries and is produced by microorganism fermentation; however, most microorganisms cannot directly utilize biomass such as starchy materials and cellulose. Here, we summarize LA production using several kinds of genetically modified microorganisms, such as LA bacteria, Escherichia coli, Corynebacterium glutamicum, and yeast. Using gene manipulation and metabolic engineering, the yield and optical purity of LA produced from biomass has been significantly improved. In this review, the drawbacks as well as improvements of LA production by fermentation is discussed.

The use of enzymes is a promising approach for site-specific protein modification on living cells owing to their substrate specificity. Herein we describe a general strategy for the site-specific modification of cell surface proteins with synthetic molecules by using Sortase, a transpeptidase from Staphylococcus aureus. The short peptide tag LPETGG is genetically introduced to the C terminus of the target protein, expressed on the cell surface. Subsequent addition of Sortase and an N-terminal triglycine-containing probe results in the site-specific labeling of the tagged protein. We were successful in the C-terminal-specific labeling of osteoclast differentiation factor (ODF) with a biotin- or fluorophore-containing short peptide on the living cell surface. The labeling reaction occurred efficiently in serum-containing medium, as well as serum-free medium or PBS. The labeled products were detected after incubation for 5 min. In addition, site-specific protein-protein conjugation was successfully demonstrated on a living cell surface by the Sortase-catalyzed reaction. This strategy provides a powerful tool for cell biology and cell surface engineering.

BackgroundThe filamentous fungus T. reesei effectively degrades cellulose and is known to produce various cellulolytic enzymes such as β-glucosidase, endoglucanase, and cellobiohydrolase. The expression levels of each cellulase are controlled simultaneously, and their ratios and synergetic effects are important for effective cellulose degradation. However, in recombinant Saccharomyces cerevisiae, it is difficult to simultaneously control many different enzymes. To construct engineered yeast with efficient cellulose degradation, we developed a simple method to optimize cellulase expression levels, named cocktail δ-integration.ResultsIn cocktail δ-integration, several kinds of cellulase expression cassettes are integrated into yeast chromosomes simultaneously in one step, and strains with high cellulolytic activity (i.e., expressing an optimum ratio of cellulases) are easily obtained. Although the total integrated gene copy numbers of cocktail δ-integrant strain was about half that of a conventional δ-integrant strain, the phosphoric acid swollen cellulose (PASC) degradation activity (64.9 mU/g-wet cell) was higher than that of a conventional strain (57.6 mU/g-wet cell). This suggests that optimization of the cellulase expression ratio improves PASC degradation activity more so than overexpression.ConclusionsTo our knowledge, this is the first report on the expression of cellulase genes by δ-integration and optimization of various foreign genes by δ-integration in yeast. This method should be very effective and easily applied for other multi-enzymatic systems using recombinant yeast.

To allow the comprehensive assessments of yeast expression systems, a simple and immediate method for simultaneously evaluating the expression level and plasmid maintenance in yeast was demonstrated. This method uses green fluorescent protein (GFP) and flow cytometry (FCM) and is characterized by a dual analysis of the average intensity of GFP fluorescence and the population of GFP-expressing cells. The FCM analysis of GFP fluorescence intensity rapidly quantifies the expression level without complex manipulations, such as the enzymatic reaction of a lacZ reporter assay. Moreover, the single-cell analysis revealed that the proportion of cells expressing GFP in the cell cluster reflects the plasmid retention rate; therefore, the FCM analysis of the GFP-expressing population allows the immediate estimation of the plasmid retention rate without the 2- or 3-day incubation required for colony counting. We show that the FCM analysis with GFP reporter is a suitable method to explore the hopeful expression vector and host strain or establish the several expression systems exhibiting the characteristic properties in yeast.