Gene Fusions for the Directed Modification of the Carotenoid Biosynthesis Pathway in Mucor circinelloides

Several fungal species, particularly some included in the Mucorales, have been used to develop fermentation processes for the production of β-carotene. Oxygenated derivatives of β-carotene are more valuable products, and the preference by the market of carotenoids from biological sources has increased the research in different carotenoid-producing organisms. We currently use
Mucor circinelloides
as a model organism to develop strains able to produce new, more valuable, and with an increased content of carotenoids. In this chapter we describe part of our efforts to construct active gene fusions which could advance in the diversification of carotenoid production by this fungus. The main carotenoid accumulated by
M. circinelloides
is β-carotene, although it has some hydroxylase activity and produces low amounts of zeaxanthin. Two enzymatic activities are required for the production of astaxanthin from β-carotene: a hydroxylase and a ketolase. We used the
ctrW
gene of
Paracoccus
sp. N81106, encoding a bacterial β-carotene ketolase, to construct gene fusions with two fungal genes essential for the modification of the pathway in
M. circinelloides
. First we fused it to the
carRP
gene of
M. circinelloides
, which is responsible for the phytoene synthase and lycopene cyclase activities in this fungus. The expected activity of this fusion gene would be the accumulation by
M. circinelloides
of canthaxanthin and probably some astaxanthin. A second construction was the fusion of the
crtW
gene of
Paracoccus
sp. to the
crtS
gene of
Xanthophyllomyces dendrorhous
, responsible for the synthesis of astaxanthin from β-carotene in this fungus, but which was shown to have only hydroxylase activity in
M. circinelloides
. The expected result in
M. circinelloides
transformants was the accumulation of astaxanthin. Here we describe a detailed and empirically tested protocol for the construction of these gene fusions.