Metabolic engineering of lactic acid bacteria for production of mannitol, L-ribulose and L-ribose

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TKK dissertations, 157
The main goal of this work was to metabolically engineer lactic acid bacteria (LAB) for the use as efficient production hosts of commercially interesting rare sugars and sugar alcohols. LAB are a very diverse group of gram positive bacteria. It is a group of bacteria that is originally identified by their ability to synthesize lactic acid. The main function of sugar metabolism in LAB is to generate the energy necessary for growth and maintenance of cell functions. Hence, their sugar metabolism is generally not connected to their limited biosynthetic activity. The possibility of modifying catabolic pathways of sugars without disturbing the biosynthesis of cell components makes the sugar metabolism of LAB attractive targets for metabolic engineering. LAB have also the GRAS (generally regarded as safe) status which makes them suitable production hosts for food and pharmaceutical applications. In the first part of this work mannitol production of Leuconostoc pseudomesenteroides was improved by random mutagenesis. Mannitol is a naturally occurring six-carbon sugar alcohol, which is widely used in the food, chemical and pharmaceutical industry. The activity of fructokinase was decreased to about 10% compared to the parent strain, which 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/mol). In addition, characteristics of fructose utilization of Lactobacillus fermentum were studied. A novel fruIK operon involved in channeling fructose to the PK pathway was characterized. In the second part of this work L-arabinose metabolism of Lactobacillus plantarum was modified. An L-ribulokinase deficient mutant was constructed. Resting cells of the ribulokinase deficient mutant were used for the production of L-ribulose. The isomerisation of L-arabinose to L-ribulose was very unfavourable for L-ribulose formation. However, high L-ribulose yields were obtained by complexing the produced L-ribulose with borate. The process for L-ribulose production in borate buffer by resting cells was optimized using central composite designs. The statistical software predicted initial L-ribulose production rate (ri) of 29.1 g/(l·h), best achievable process productivity (rmax) of 14.8 g/(l·h) and conversion of L-arabinose to L-ribulose (x) of 0.70 mol/mol. The work was continued by introducing L-ribose isomerase activity into L-ribulokinase deficient strains of E. coli and Lb. plantarum. By adding the second reaction, isomerization of L-ribulose to L-ribose, the two-reaction sequence with L-arabinose as the starting material became favorable for L-ribose production. L-Ribose is a rare sugar that is used as a precursor for a production of antiviral drugs. The process for L-ribose production by resting cells and protein precipitates was investigated. The initial L-ribose production rates were 0.46 g/(g·h) [1.8 g/(l·h)] and 0.27 g/(g·h) [1.91 g/(l·h)] for E. coli and for Lb. plantarum, respectively. Conversions were around 20% (mol/mol) at their highest in the experiments.
lactic acid bacteria, mannitol, L-ribulose, L-ribose
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  • [Publication 1]: Miia Helanto, Johannes Aarnikunnas, Niklas von Weymarn, Ulla Airaksinen, Airi Palva, and Matti Leisola. 2005. Improved mannitol production by a random mutant of Leuconostoc pseudomesenteroides. Journal of Biotechnology, volume 116, number 3, pages 283-294.
  • [Publication 2]: Miia Helanto, Johannes Aarnikunnas, Airi Palva, Matti Leisola, and Antti Nyyssölä. 2006. Characterization of genes involved in fructose utilization by Lactobacillus fermentum. Archives of Microbiology, volume 186, number 1, pages 51-59.
  • [Publication 3]: M. Helanto, K. Kiviharju, M. Leisola, and A. Nyyssölä. 2007. Metabolic engineering of Lactobacillus plantarum for production of L-ribulose. Applied and Environmental Microbiology, volume 73, number 21, pages 7083-7091. © 2007 American Society for Microbiology (ASM). By permission.
  • [Publication 4]: M. Helanto, K. Kiviharju, T. Granström, M. Leisola, and A. Nyyssölä. 2009. Biotechnological production of L-ribose from L-arabinose. Applied Microbiology and Biotechnology, in press. Published online 17th of January 2009.