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A mutant of Leuconostoc pseudomesenteroides ATCC12291 that was unable to grow on fructose was constructed by chemical mutagenesis. The fructose uptake of this mutant, designated as BPT143, was unaltered and allowed fructose still to be converted into mannitol when glucose was present in the growth medium. The mutant grew and consumed fructose faster than the parent strain when grown in a medium containing both glucose and fructose. The specific activity of fructokinase, the enzyme involved in phosphorylation of fructose to fructose-6-phosphate, was decreased to about 10% of that of the parent strain, and resulted in a reduced leakage of fructose into the phosphoketolase (PK) pathway. The yield of mannitol from fructose was improved from 74 to 86 mol%. The increased fructose consumption rate and higher mannitol yield of the mutant also resulted in improvement of volumetric mannitol productivity. In addition, isolation and characterization of the wild type L. pseudomesenteroides fructokinase gene (fruK) was performed. DNA sequence analysis of the fruK gene region of BPT143 revealed only one silent mutation which does not explain the highly reduced fructokinase activity of the mutant. The genetic characterization of fruK was completed by analyzing the expression, size and 5' end of fruK transcripts. Expression data with BPT143, revealing absence of fruK transcripts, was in accordance with the reduced fructokinase activity of the mutant.

For production of mannitol in combination with pure L-lactic acid or pyruvate, the D- and L-lactate dehydrogenase genes (ldhD and ldhL) of a mannitol-producing Lactobacillus fermentum strain were cloned and stepwise inactivated. For inactivation of both ldh genes by a gene replacement technique, deletion constructs removing a 0.4-kb fragment from the promoter and the 5' end region of the ldh genes were used. The first inactivation mutant, designated L. fermentum GRL1030, carried the deletion in ldhD (DeltaldhD). A double mutant, DeltaldhD-DeltaldhL, was constructed by the inactivation of the ldhL gene of strain GRL1030, resulting in strain L. fermentum GRL1032. The correctness of the both mutants was confirmed at the DNA level by polymerase chain reaction, as shown by the absence of ldh transcripts by northern blotting and as a lack of the corresponding enzyme activity. In bioreactor cultivations, the single mutant GRL1030 produced mannitol and L-lactic acid as expected. Mannitol and lactic acid yields and productivities were practically unaffected by deletion of the ldhD gene. The double mutant GRL1032 produced mannitol and pyruvate as expected. However, although the yield of mannitol from fructose remained high, its volumetric productivity was reduced. The double mutation negatively affected the glucose consumption rate, resulting in reduced cellular growth. In addition to pyruvate, the double mutant produced 2,3-butanediol. More surprisingly, some lactic acid was still produced., (Copyright 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 82: 653-663, 2003.)

D-Mannitol is a sugar alcohol with applications in chemistry, food and pharmaceutical industries, and medicine. Commercially, mannitol is produced by catalytic hydrogenation. Although this process is widely used, it is not optimal for mannitol production. New processes, including chemical, enzymatic, and microbial processes, are frequently developed and evaluated against the existing hydrogenation processes. In earlier papers, we have described the identification of a food-grade lactic acid bacterium strain, Leuconostoc mesenteroides ATCC-9135, with efficient mannitol production capabilities and the development and optimization of a new bioprocess in which the strain was applied. The new bioprocess is simple. It requires a reduced bioreactor with the following features: sterilization, pH and T control (at mild conditions), and slow mixing. The contamination risk of the new bioprocess is low, and the downstream processing protocol comprises simple, widely used unit operations: evaporation, crystallization, crystal separation, and drying. On a 2-L laboratory scale, high mannitol yields from fructose (93-97%) and volumetric mannitol productivities (>20 g L(-1) h(-1)) were achieved. In this paper, the scalability of the new bioprocess was tested on a small pilot scale (100 L). In the pilot plant, production levels were achieved similar to those in the laboratory. Also, high-purity mannitol crystals were obtained at similar yield levels. The results presented in this paper indicate that the new bioprocess can easily be scaled-up to an industrial scale and that the production levels achieved with it are comparable to the catalytic hydrogenation processes.

Eight heterofermentative lactic acid bacteria were compared as to their ability to convert d-fructose to d-mannitol. Four promising strains were identified and the effects of growth temperature, pH, and nitrogen flushing on mannitol production by these strains were studied in batch bioreactor cultivations. In contrast to earlier findings, a high growth temperature was observed to improve the yield of mannitol from fructose with Lactobacillus fermentum. When Lb. fermentum was grown at 25, 30, and 35 °C, yields of 86.4±0.8, 88.9±2.4, and 93.6±0.6 mol%, respectively, were achieved. In general, constant nitrogen gas flushing of the growth media was found to improve the mannitol yields, but not the volumetric mannitol productivities. Applying the most promising strain in a batch mannitol production experiment, high average and maximum volumetric mannitol productivities (7.6 and 16.0 g/l/h, respectively) were achieved. [Copyright &y& Elsevier]