Biosynthesis of Poly(3-hydroxyalkanoate) from Amino Acids in Medium with Nitrogen, Phosphate, and Magnesium, or Some Combination of These Nutrients

Abstract

Twenty natural amino acids were investigated as carbon sources for biosynthesis of poly(3-hydroxyalkanoate) (PHA) by Ralstonia eutropha in media free of inorganic nitrogen, phosphate, or magnesium. First, the effect of limiting nitrogen, phosphate, and magnesium was investigated on the metabolism of l-leucine. Nitrogen-limited media have been widely used to stimulate PHA accumulation, but phosphate-free media lead to higher accumulation. This is because amino acids can act as nitrogen sources, leading to preferential cell growth over PHA accumulation. Magnesium-free conditions don’t show a significant effect on accumulation of PHA. When Ralstonia eutropha was cultivated in the presence of natural amino acids l-leucine, l-isoleucine, l-phenylalanine, and l-tyrosine in media free of nitrogen, phosphate, and magnesium, the PHA content was high, over 40 % of dry weight. Accumulation of PHA on supplementation with mixed substrates of l-leucine and various other amino acids was investigated in nitrogen-, phosphate-, and magnesium-free medium. Culturing with most mixed substrates led to accumulation of PHA, but some led to low or no PHA yield in spite of high PHA yield when metabolized from l-leucine alone. l-cysteine as a sole carbon source showed a unique feature, in that cell growth was significantly preferred over PHA accumulation. A mixed substrate of l-leucine and l-cysteine provided high PHA accumulation because of the combination of PHA accumulation due to l-leucine and cell growth due to l-cysteine. When glucose was used instead of l-leucine in a mixed substrate with l-cysteine, the PHA content was much lower because l-cysteine acts as an inhibitor of glucose metabolism. These results showed that the precise combination of carbon sources is an important factor in accumulation of PHA.