Cholesterol oxidases are important enzymes with a wide range of applications from basic research to industry. In this study, we have discovered and described the first cell-associated cholesterol oxidase, ChoD, from
Streptomyces lavendulae
YAKB-15. This strain is a naturally high producer of ChoD, but only produces ChoD in a complex medium containing whole yeast cells. For characterization of ChoD, we acquired a draft genome sequence of
S
.
lavendulae
YAKB-15 and identified a gene product containing a flavin adenine dinucleotide binding motif, which could be responsible for the ChoD activity. The enzymatic activity was confirmed
in vitro
with histidine tagged ChoD produced in
Escherichia coli
TOP10, which lead to the determination of basic kinetic parameters with
K
m
15.9 µM and
k
cat
10.4/s. The optimum temperature and pH was 65 °C and 5, respectively. In order to increase the efficiency of production, we then expressed the cholesterol oxidase,
choD
, gene heterologously in
Streptomyces lividans
TK24 and
Streptomyces albus
J1074 using two different expression systems. In
S
.
albus
J1074, the ChoD activity was comparable to the wild type
S
.
lavendulae
YAKB-15, but importantly allowed production of ChoD without the presence of yeast cells.
Actinomycetes are important producers of pharmaceuticals and industrial enzymes. However, wild type strains require laborious development prior to industrial usage. Here we present a generally applicable reporter-guided metabolic engineering tool based on random mutagenesis, selective pressure, and single-cell sorting. We developed fluorescence-activated cell sorting (FACS) methodology capable of reproducibly identifying high-performing individual cells from a mutant population directly from liquid cultures. Genome-mining based drug discovery is a promising source of bioactive compounds, which is complicated by the observation that target metabolic pathways may be silent under laboratory conditions. We demonstrate our technology for drug discovery by activating a silent mutaxanthene metabolic pathway in Amycolatopsis. We apply the method for industrial strain development and increase mutaxanthene yields 9-fold to 99 mg l-1 in a second round of mutant selection. Actinomycetes are an important source of catabolic enzymes, where product yields determine industrial viability. We demonstrate 5-fold yield improvement with an industrial cholesterol oxidase ChoD producer Streptomyces lavendulae to 20.4 U g-1 in three rounds. Strain development is traditionally followed by production medium optimization, which is a time-consuming multi-parameter problem that may require hard to source ingredients. Ultra-high throughput screening allowed us to circumvent medium optimization and we identified high ChoD yield production strains directly from mutant libraries grown under preset culture conditions. In summary, the ability to screen tens of millions of mutants in a single cell format offers broad applicability for metabolic engineering of actinomycetes for activation of silent metabolic pathways and to increase yields of proteins and natural products.
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