Abstract
D-lactate is a natural component of many fermented foods like yogurts, sour milk, cheeses, and pickles vegetable products. D-lactate in high concentrations is toxic for children and people with short bowel syndrome and provokes encephalopathy. These facts convincingly demonstrate a need for effective tools for the D-lactate removal from some food products. The main idea of investigation is focused on application of recombinant thermotolerant methylotrophic yeast Hansenula polymorpha “tr6,” overproducing D-lactate: cytochrome c oxidoreductase (EC 1.1.2.4, D-lactate cytochrome c oxidoreductase, D-lactate dehydrogenase (cytochrome), DLDH). In addition to 6-fold overexpression of DLDH under a strong constitutive promoter (prAOX), the strain of H. polymorpha “tr6” (gcr1 catX/Δcyb2, prAOX_DLDH) is characterized by impairment in glucose repression of AOX promoter, devoid of catalase and L-lactate-cytochrome c oxidoreductase activities. Overexpression of DLDH coupling with the deletion of L-lactate-cytochrome c oxidoreductase activity opens possibility for usage of the strain as a base for construction of bioreactor for removing D-lactate from fermented products due to oxidation to nontoxic pyruvate. A laboratory prototype of column-type bioreactor for removing a toxic D-lactate from model solution based on permeabilized cells of the H. polymorpha “tr6” and alginate gel was constructed and efficiency of this process was tested.
Highlights
D-lactate is a natural component of many fermented foods like yogurts [1, 2], sour milk [3], cheeses [4], and pickles vegetable products [5].D-lactate can be metabolized slowly in the human body in comparison with L-lactate isomer and can cause metabolic disorders if ingested in excess
We propose using recombinant DLDH-overproducing strain H. polymorpha “tr6” as a base for construction of laboratory prototype of bioreactor for removing a toxic D-lactate from fermented products
The gene CYB2 was deleted on the background of the b-105 strain of H. polymorpha impaired in glucose repression and devoid of catalase activity to avoid specific L-lactate-cytochrome c oxidoreductase activity
Summary
D-lactate is a natural component of many fermented foods like yogurts [1, 2], sour milk [3], cheeses [4], and pickles vegetable products [5].D-lactate can be metabolized slowly in the human body in comparison with L-lactate isomer and can cause metabolic disorders if ingested in excess. D-lactate is a natural component of many fermented foods like yogurts [1, 2], sour milk [3], cheeses [4], and pickles vegetable products [5]. Some countries, for example, Germany, have decided to minimize the D-lactate content of fermented dairy products [6]. The high concentration of D-lactate is toxic for children [7] and people with short bowel syndrome [8,9,10]. D-lactate toxicity depends on physiological state of renal excretion. In 1993, there was reported a case of chronic D-lactate encephalopathy occurring in a patient with short bowel syndrome and end-stage renal disease [12]
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