Effect of expressing polyhydroxybutyrate synthesis genes (phbCAB) in Streptococcus zooepidemicus on production of lactic acid and hyaluronic acid.
Hyaluronic acid (HA) has been industrially produced using the gram-positive bacterium Streptococcus zooepidemicus. Large amount of lactic acid formation was one of the important factors that restricted cell growth and HA productivity and lowered the substrate to HA conversion efficiency in a fermentor. In this study, polyhydroxybutyrate (PHB) synthesis genes (phbCAB) of Ralstonia eutropha were cloned from the plasmid pBHR68 and were inserted into the plasmid pEU308, an expression vector for gram-positive bacteria. The plasmid was transformed into S. zooepidemicus by electroporation. beta-Ketothiolase (PhbA), acetoacetyl-CoA reductase (PhbB), and polyhydroxyalkanoate (PHA) synthase (PhbC) activity assays were carried out to demonstrate the expression of these genes. The PhbA and PhbB activities were 3.13 and 1.23 U mg(-1), respectively. No PhbC activities were detected. In shake flask studies, there was no obvious difference between the wild-type and recombinant S. zooepidemicus harboring phbCAB genes in terms of lactic acid and HA formation. However, in fermentor studies, the recombinant produced only 40 g L(-1) lactic acid and 7.5 g L(-1) HA, whereas the wild type produced 65 g L(-1) lactic acid and 5.5 g L(-1) HA. These results suggested that expression of phbCAB genes in S. zooepidemicus could help regulate HA production metabolism. Because the lactic acid formation in S. zooepidemicus was sensitive to cellular oxidation/reduction potential, it is proposed that the PHB synthesis pathway could act as a regulator to adjust the cellular oxidation/reduction potential. This is the first study demonstrating that PHA synthesis related to energy and carbon metabolism could be employed as a pathway to regulate other cellular metabolism and possibly to regulate the production of other metabolic products.
Zhang
Jinyu
J
Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing, China.
Hao
Ning
N
Chen
Guo-Qiang
GQ
D000102:Acetyl-CoA C-Acyltransferase / Q000235:genetics / Q000378:metabolism; D000217:Acyltransferases / Q000235:genetics / Q000378:metabolism; D000429:Alcohol Oxidoreductases / Q000235:genetics / Q000378:metabolism; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D006820:Hyaluronic Acid / Q000096:biosynthesis*; D006885:Hydroxybutyrates / Q000378:metabolism*; D019344:Lactic Acid / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D018502:Streptococcus equi / Q000235:genetics / Q000378:metabolism*; D014170:Transformation, Genetic
D004269:DNA, Bacterial; D006885:Hydroxybutyrates; D019344:Lactic Acid; D006820:Hyaluronic Acid; D000429:Alcohol Oxidoreductases; C023744:acetoacetyl-CoA reductase; D000217:Acyltransferases; C077225:poly(3-hydroxyalkanoic acid) synthase; D000102:Acetyl-CoA C-Acyltransferase