METABOLIC ENGINEERING OF SERRATIA-MARCESCENS WITH THE BACTERIAL HEMOGLOBIN GENE - ALTERATIONS IN FERMENTATION PATHWAYS

Serratia marcescens was transformed with plasmid vector pUC8 or pUC8 c ontaining the bacterial (Vitreoscilla) hemoglobin gene (vgb) on either a 2.3-kb fragment (pUC8:15) or 1.4-kb fragment (pUC8:16) of Vitreosci lla DNA. The vgb-bearing strains were compared with the pUC8 transform ant and untransformed S. marcescens with respect to growth in Luria-Be rtani (LB) broth supplemented with glucose or casein acid hydrolysate. Growth (on a viable cell basis) was similar to that in unsupplemented LB. Total acid excretion (as estimated by medium pH) was similar for all strains in both LB plus 2% casein acid hydrolysate and LB without additions. Acid excretion in LB plus 2% glucose was somewhat greater a t up to 10 h in culture for the two vgb-bearing strains; from 10 to 26 h in culture, the pHs of these cultures continued to decrease (to 4.1 -4.2), whereas those of the non-vgb-bearing strains returned to near t he starting pH (7.4-7.8). Concomitantly, after 26 h of culture in LB p lus 2% glucose, the non-vgb-bearing strains had produced about 15 time s as much acetoin and about three to four times as much 2,3-butanediol as the vgb-bearing strains. In general, for all strains, much more ac etoin and 2,3-butanediol were produced in LB plus 2% glucose than in u nsupplemented LB. The exception was acetoin production by the strain b earing vgb on plasmid pUC8:15; after 26 h of culture in LB without sup plementation it was between three and four times that of the other str ains, and about 50% higher than its level in LB plus 2% glucose. When grown with the 2% casein acid hydrolysate supplement, the strain beari ng vgb on plasmid pUC8:15 produced much more acetoin and 2,3-butanedio l than the other strains after 26 hours in culture. The results confir m that vgb can significantly alter carbon metabolism and suggest that the use of vgb technology for directed metabolic engineering may be a complicated process, depending in part on medium composition. (C) 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59: 640-646,1998.