http://www.sklbpi.cn/jinqifabiaolunwen/749.html
Abstract
Background: Milbemycins, a
group of 16-membered macrolides with potent anthelminthic and insecticidal
activity,are produced by several Streptomyces and used widely in agricultural,
medical and veterinary fields. Milbemycin A3 and A4, the main components
produced by Streptomyces bingchenggensis, have been developed as an acaricide to
control mites. The subsequent structural modification of milbemycin A3/A4 led to
other commercial products, such as milbemycin oxime, lepimectin and latidectin.
Despite its importance, little is known about the regulation of milbemycin
biosynthesis, which has hampered efforts to enhance milbemycin production via
engineering regulatory genes.
Results: milR, a regulatory gene in the
milbemycin (mil) biosynthetic gene cluster of S. bingchenggensis, encodes a
large ATP-binding regulator of the LuxR family (LAL family), which contains an
ATPase domain at its N-terminus and a LuxR-like DNA-binding domain at the
C-terminus. Gene disruption and genetic complementation revealed that milR plays
an important role in the biosynthesis of milbemycin. β-glucuronidase assays and
transcriptional analysis showed that MilR activates the expression of the
milA4-E operon and milF directly, and activates the other mil genes indirectly.
Site-directed mutagenesis confirmed that the ATPase domain is indispensable for
MilR’s function, and particularly mutation of the conserved amino acids K37A,
D122A and D123A, led to the loss of MilR function for
milbemycin
biosynthesis. Overexpression of an extra copy of milR under the
control of its native promoter significantly increased production of milbemycin
A3/A4 in a high-producing industrial strain S. bingchenggensis BC04.
Conclusions: A LAL regulator, MilR, was characterized in the mil gene cluster of
S. bingchenggensis BC04. MilR could activate milbemycin biosynthesis through
direct interaction with the promoter of the milA4-E operon and that of milF.
Overexpression of milR increased milbemycin A3/A4 production by 38 % compared
with the parental strain BC04, suggesting that genetic manipulation of this
activator gene could enhance the yield of antibiotics.
Keywords: Milbemycin,
LAL, MilR, Overexpression, Streptomyces bingchenggensis BC04.
