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Microorganisms
2019 Aug 28;79:. doi: 10.3390/microorganisms7090294.
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Heterologous Hyaluronic Acid Production in Kluyveromyces lactis.
V Gomes AM
,
C M Netto JH
,
Carvalho LS
,
Parachin NS
.
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Hyaluronic Acid (HA) is a biopolymer composed by the monomers Glucuronic Acid (GlcUA) and N-Acetyl Glucosamine (GlcNAc). It has a broad range of applications in the field of medicine, being marketed between USD 1000-5000/kg. Its primary sources include extraction of animal tissue and fermentation using pathogenic bacteria. However, in both cases, extensive purification protocols are required to prevent toxin contamination. In this study, aiming at creating a safe HA producing microorganism, the generally regarded as safe (GRAS) yeast Kluyveroymyces lactis is utilized. Initially, the hasB (UDP-Glucose dehydrogenase) gene from Xenopus laevis (xlhasB) is inserted. After that, four strains are constructed harboring different hasA (HA Synthase) genes, three of humans (hshasA1, hshasA2, and hshasA3) and one with the bacteria Pasteurella multocida (pmhasA). Transcript values analysis confirms the presence of hasA genes only in three strains. HA production is verified by scanning electron microscopy in the strain containing the pmHAS isoform. The pmHAS strain is grown in a 1.3 l bioreactor operating in a batch mode, the maximum HA levels are 1.89 g/L with a molecular weight of 2.097 MDa. This is the first study that reports HA production in K. lactis and it has the highest HA titers reported among yeast.
Figure 1. The metabolic pathway for Hyaluronic Acid production. In green, the enzymes that are not present in the metabolism of K. lactis but are necessary for the synthesis of Hyaluronic Acid.
Figure 2. Overall construction strategy for the four strains generated in this study. The hasB gene from Xenopus laevis (xlhasB) and the different versions of hasA genes from Homo sapiens (hshasA1, hshasA2, and hshasA3) and Pasteurella multocida (pmhasA) were inserted into the integrative plasmid pKlac2 for recombination in the LAC4 promoter of the K. lactis genome.
Figure 3. Scanning electron microscopy (SEM) analysis of the wild-type K. lactis GG799 strain and all strains constructed in this study. (a) GG799 strain (6000-fold increase) after 24 h of flask cultivation; (b) BA1 strain (5000-fold increase) after 24 h of flask cultivation; (c) BA2 strain (5000-fold increase) after 24 h of flask cultivation; (d) BA3 strain (5000-fold increase) after 24 h of flask cultivation; (e) BAP strain (5000-fold and 15000-fold increase) after 6 h of flask cultivation and (f) BAP strain after 24 h of flask cultivation in 5000-fold increase.
Figure 4. Gel Permeation Chromatography (GPC) analysis of the Hyaluronic Acid (HA) purified from BAP strain after growth in bioreactor.
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