초록
<P><B>Abstract</B></P> <P>In this study, an astaxanthin-biosynthesis <I>Kluyveromyces marxianus</I> strain Sm23 was first constructed, which could produce 31µg/g DCW astaxanthin. Then, repeated genome integration of the key astaxanthin biosynthesis genes <I>Hpchyb</I> and <I>bkt</I> was done to increase gene copy number and astaxanthin yield. Four improved strains were obtained and the yield of astaxanthin and the total yield of carotenoids in a strain increased with the copy numbers of <I>Hpchyb</I> and <I>bkt</I>. To improve the yield further, the gene <I>Hpchyb</I> from <I>Haematococcus pluvialis</I> was modified by site-directed mutagenesis to increase the enzyme efficiency or/and to prevent the heterologous protein degradation by ubiquitination. Using repeated-integration approach of <I>bkt</I> and the mutated <I>Hpchyb</I> into Sm23, the S3-2 strain was obtained and shown to produce the 3S, 3′S-astaxanthin at 9972µg/g DCW in a 5L fermentor.</P> <P><B>Highlights</B></P> <P> <UL> <LI> An astaxanthin-biosynthesis pathway was constructed in <I>Kluyveromyces marxianus</I>. </LI> <LI> Repeated genome integration of <I>bkt</I> and <I>Hpchyb</I> increased astaxanthin yield. </LI> <LI> <I>Hpchyb</I> was modified by site-directed mutagenesis to improve the enzyme activity. </LI> <LI> Our newly constructed strain can produce 3S, 3′S-astaxanthin at a high rate. </LI> </UL> </P>