A R T I C L E S
Pickens et al.
chlorothricin28 (ChlJ), jadomycin40 (JadN), simocyclinone24
(SimA12), medermycin41 (med-ORF22), and others. One pos-
sible role for SsfE is to synthesize methylmalonyl-CoA 22 from
21. The next set of steps in the biosynthesis of 25 are proposed
to be catalyzed by ssfK, ssfN, and ssfJ, which are cotranscribed
as a tricistronic cassette on the same operon. SsfN is homologous
to KS III enzymes such as FabH, which catalyzes the condensa-
tion between acetyl-CoA and malonyl-ACP to initiate fatty acid
biosynthesis.42 FabH homologues have been found in a number
of polyketide gene clusters such as in R1128,43 frenolicin,43
and daunorubicin44 to catalyze the decarboxylative condensation
of short acyl groups. SsfN may therefore catalyze the condensa-
tion of 22 with acetyl-CoA to form 2-methyl-acetoacetyl-CoA
23. ꢀ-Ketoreduction of 23 catalyzed by SsfK, which is
homologous to 3-oxoacyl-ACP reductases such as FabG,45
yields 3-hydroxyl-2-methyl-butyryl-CoA 24. Stereospecific de-
hydration of 24 by SsfJ, which is a member of the enoyl-CoA
hydratase/isomerase family (PF00378), results in 25. As an
alternative start to this pathway, SsfN may directly condense
malonyl-CoA with acetyl-CoA to form acetoacetyl-CoA. The
R-methyl group may then be incorporated by SsfM3, which is
homologous to C-methyltransferases, to afford 23.
Transfer of 25 to 7 is predicted to be catalyzed by an
acyltransferase using similar mechanisms as for other acylated
deoxysugars present in chromomycin46 or mannopeptimycin.47
Those belong to a large acyltransferase family (PF01757) that
contains proteins with O-acetyltransferase activity, including the
deoxyhexose O-acyltransferases MdmB.48 Unexpectedly, no
similar acyltransferase is present in the ssf gene cluster. One
acyltransferase homologue, SsfV, however, remains unassigned.
SsfV has homologues in many type II polyketide gene clusters
including OxyP from the oxy pathway and ZhuC from the R1128
pathway. A Pfam homology search revealed that it contains an
acyltransferase domain (PF00698) from a family of proteins that
includes bacterial malonyl-CoA-ACP transacylase (MAT). In
vitro experiments with homologue ZhuC showed that it did
indeed have malonyl-CoA ACP transacylase activity, however
it was much slower than endogenous MAT from the fatty acid
biosynthetic pathway.49,50 Additional in vitro studies demon-
strated that ZhuC could efficiently hydrolyze acetyl and pro-
pionyl primed ACPs and therefore acts to ensure that the R1128
PKS is primed with the correct medium chain length acyl-
ACPs.50 Similarly, SsfV may be involved in ensuring the
biosynthesis of 1 is initiated with malonamyl starter unit and is
hence unlikely to be involved in angelate attachment.
biosynthetic cluster.28 The role of these genes in the processing
and attachment of the 5-chloro-6-methyl-O-methylsalicylic acid
during chlorothricin biosynthesis has recently been published.51
ChlB3 is utilized to transfer 6-methylsalicylic acid from the type
I PKS ChlB1 to a discrete ACP for further tailoring. ChlB6 is
shown to transfer the tailored 5-chloro-6-methyl-O-methylsali-
cylic acid to D-olivose of desmethylsalicyl chlorothricin to form
chlorothricin.51 Considering SsfG displays high sequence simi-
larity to ChlB6 (52%) and the same deoxysugar substrate
D-olivose is involved, SsfG therefore likely catalyzes the
attachment of angelate in the ssf pathway. Instead of using a
discrete ACP as the acyl carrier, the CoA-activated angelate 25
may directly serve as the substrate of SsfG.
Genes Encoding Enzymes That Synthesize the Polyketide
Backbone 40. The ssf minimal PKS consists of SsfA (KSR), SsfB
(KSꢀ or CLF), and SsfC (ACP) that are highly homologous to
the oxy minimal PKS.18 The amidotransferase SsfD, which is
responsible for producing the malonamate starter unit unique
to tetracyclines, is found adjacent to the ssf minimal PKS. The
C-2 amidated starter unit is one of the signature moieties of
tetracyclines, and the revelation of SsfD as an OxyD homologue
with 68% sequence identity was one of the earliest convincing
pieces of evidence that this gene cluster indeed encodes a
tetracycline compound. Like OxyD, SsfD is highly similar to
class II asparagine synthases that utilize ATP to convert aspartate
to asparagine using glutamine as the nitrogen source.52 Zhang
and co-workers demonstrated that the “extended minimal PKS”,
which includes OxyABCD, is sufficient for producing the
amidated polyketide chain 38.18 In addition, SsfU, which is 76%
identical to OxyJ, is predicted to be the C-8 (or C-9* using
biosynthetic carbon numbering as shown in Scheme 4) ketore-
ductase that regioselectively reduces the nascent backbone to
yield 40.20
To verify the function of the ssf minimal PKS, genes encoding
SsfA, SsfB and SsfC, SsfD, and SsfU were placed into a pRM5-
derived Streptomyces-E. coli shuttle vector53 (Table 2). Shuttle
plasmid pLP27 was subsequently transformed into S. liVidans
strain K4-114.54 The resulting organic extract was analyzed by
HPLC and is shown in Figure 5A. The product profile of K4-
114/pLP27 was indistinguishable from that of K4-114/pWJ35,
which contained the corresponding enzymes from the oxy
biosynthetic pathway.18 By comparison to an authentic standard,
the major product of the extract (RT ) 13.5 min) was confirmed
to be the isoquinolone 42 (20 mg/L), which can form via the
spontaneous cyclization of the C-9* reduced amidated polyketide
40.18 We further investigated whether the oxy and ssf extended
minimal PKS components are functionally interchangeable.
Three additional shuttle vectors were prepared with a combina-
tion of oxy and ssf genes as shown in Table 2 and transformed
into K4-114. Each of these host/vector combinations was
capable of producing 42 in similar quantities as K4-114/pLP27,
indicating that each of the ssf components is functionally
compatible and equivalent to its oxy counterpart. These results
confirm (i) the extended ssf minimal PKS (SsfABCD) is capable
of synthesizing the full length amidated polyketide precursor
38, (ii) the hypothesis that the carbon backbone of 1 is
The ssf gene cluster encodes another KS III homologue SsfG,
which resembles ChlB3 and ChlB6 from the chlorothricin
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