Metabolic engineering of TZ1 for improved malic acid production.

RK089 has been found recently as a good natural malic acid producer from glycerol. This strain has previously undergone adaptive laboratory evolution for enhanced substrate uptake rate resulting in the strain TZ1. Medium optimization and investigation of process parameters enabled titers and rates that are able to compete with those of organisms overexpressing major parts of the underlying metabolic pathways. Metabolic engineering can likely further increase the efficiency of malate production by this organism, provided that basic genetic tools and methods can be established for this rarely used and relatively obscure species. Here we investigate and adapt existing molecular tools from for use in . Selection markers from that confer carboxin, hygromycin, nourseothricin, and phleomycin resistance are applicable in . A plasmid was constructed containing the -locus of RK089, resulting in site-specific integration into the genome. Using this plasmid, overexpression of pyruvate carboxylase, two malate dehydrogenases (, ), and two malate transporters (, ) was possible in TZ1 under control of the strong P promoter. Overexpression of , , , and increased the product (malate) to substrate (glycerol) yield by up to 54% in shake flasks reaching a titer of up to 120 g L. In bioreactor cultivations of TZ1 P and TZ1 P a drastically lowered biomass formation and glycerol uptake rate resulted in 29% (Ssu1) and 38% (Mdh2) higher specific production rates and 38% (Ssu1) and 46% (Mdh2) increased yields compared to the reference strain TZ1. Investigation of the product spectrum resulted in an 87% closed carbon balance with 134 g L malate and biomass (73 g L), succinate (20 g L), CO (7 g L), and α-ketoglutarate (8 g L) as main by-products. These results open up a wide range of possibilities for further optimization, especially combinatorial metabolic engineering to increase the flux from pyruvate to malic acid and to reduce by-product formation.