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The bacterial hemoglobin from Vitreoscilla has been shown to increase growth yield and yield of genetically engineered product in Escherichia coli. To test the generality of this phenomenon, the approximately 560-bp bacterial (Vitreoscilla) hemoglobin gene (vgb) (including the native promoter), cloned into the vector pUC8 in two constructs containing about 1650 and 850 bp, respectively, of Vitreoscilla DNA downstream of vgb, was transformed into Serratia marcescens. After several transfers of the transformants on selective media, both plasmids became stable in this host and the resulting strains produced hemoglobin. Both transformants were compared, regarding growth in liquid Luria-Bertani (LB) medium, with untransformed S. marcescens and S. marcescens transformed with pUC8. The vgb-bearing strains had about 5 times lower maximum viable cell numbers than the strains without hemoglobin, but the former also had late log or early stationary phase cells that were 5-10 times larger than those of the latter. Further, on a dry cell mass basis the presence of vgb inhibited cell growth in liquid media. In contrast, growth of the vgb-bearing strains on LB plates based on cell mass (determined from colony size) was markedly enhanced compared with that of the pUC8 transformant. Respiration of the vgb-bearing strains was lower than that of the strains without vgb on a cell mass basis. These results show that the presence of vgb can have idiosyncratic effects and is not always an aid to cell growth so that its use for genetic engineering must be tested on a case by case basis.

Serratia marcescens was transformed with plasmid vector pUC8 or pUC8 containing the bacterial (Vitreoscilla) hemoglobin gene (vgb) on either a 2.3-kb fragment (pUC8:15) or 1.4-kb fragment (pUC8:16) of Vitreoscilla DNA. The vgb-bearing strains were compared with the pUC8 transformant and untransformed S. marcescens with respect to growth in Luria-Bertani (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 at 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 the starting pH (7.4-7.8). Concomitantly, after 26 h of culture in LB plus 2% glucose, the non-vgb-bearing strains had produced about 15 times 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 acetoin and 2,3-butanediol were produced in LB plus 2% glucose than in unsupplemented LB. The exception was acetoin production by the strain bearing vgb on plasmid pUC8:15; after 26 h of culture in LB without supplementation it was between three and four times that of the other strains, and about 50% higher than its level in LB plus 2% glucose. When grown with the 2% casein acid hydrolysate supplement, the strain bearing vgb on plasmid pUC8:15 produced much more acetoin and 2,3-butanediol than the other strains after 26 hours in culture. The results confirm 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.

Synthesis and excretion of alpha-amylase is investigated in batch cultures of Escherichia coli JM103[pMK57] (vgb-) and E. coli JM103[pMK79] (vgb+). While total production and excretion of alpha-amylase were promoted in Luria broth (LB) (excretion being as high as 87%), cell-mass-specific production of the enzyme was promoted in M9 in bioreactor cultures and in LB in shake flask cultures. Low aeration and agitation rates and presence of starch were conducive to alpha-amylase synthesis in E. coli JM103[pMK79]. Two-stage bioreactor operating strategies that will improve alpha-amylase production are proposed. The potential of these strategies is demonstrated via two-stage shake flask cultures.

Xanthomonas maltophilia was transformed with the gene encoding Vitreoscilla (bacterial) hemoglobin, vgb, and the growth of the engineered strain was compared with that of the untransformed strain using benzoic acid as the sole carbon source. In general, growth of the engineered strain was greater than that of the untransformed strain; this was true for experiments using both overnight cultures and log phase cells as inocula, but particularly for the latter. In both cases the engineered strain was also more efficient than the untransformed strain in converting benzoic acid into biomass.