초록
<P><B>Background</B></P><P>The xanthophyll astaxanthin is a high-value compound with applications in the nutraceutical, cosmetic, food, and animal feed industries. Besides chemical synthesis and extraction from naturally producing organisms like <I>Haematococcus pluvialis</I>, heterologous biosynthesis in non-carotenogenic microorganisms like <I>Escherichia coli</I>, is a promising alternative for sustainable production of natural astaxanthin. Recent achievements in the metabolic engineering of <I>E. coli </I>strains have led to a significant increase in the productivity of carotenoids like lycopene or β-carotene by increasing the metabolic flux towards the isoprenoid precursors. For the heterologous biosynthesis of astaxanthin in <I>E. coli</I>, however, the conversion of β-carotene to astaxanthin is obviously the most critical step towards an efficient biosynthesis of astaxanthin.</P><P><B>Results</B></P><P>Here we report the construction of the first plasmid-free <I>E. coli </I>strain that produces astaxanthin as the sole carotenoid compound with a yield of 1.4 mg/g cdw (<I>E. coli </I>BW-ASTA). This engineered <I>E. coli </I>strain harbors xanthophyll biosynthetic genes from <I>Pantoea ananatis </I>and <I>Nostoc punctiforme </I>as individual expression cassettes on the chromosome and is based on a β-carotene-producing strain (<I>E. coli </I>BW-CARO) recently developed in our lab. <I>E. coli </I>BW-CARO has an enhanced biosynthesis of the isoprenoid precursor isopentenyl diphosphate (IPP) and produces β-carotene in a concentration of 6.2 mg/g cdw. The expression of <I>crtEBIY </I>along with the β-carotene-ketolase gene <I>crtW148 </I>(NpF4798) and the β-carotene-hydroxylase gene (<I>crtZ</I>) under controlled expression conditions in <I>E. coli </I>BW-ASTA directed the pathway exclusively towards the desired product astaxanthin (1.4 mg/g cdw).</P><P><B>Conclusions</B></P><P>By using the λ-Red recombineering technique, genes encoding for the astaxanthin biosynthesis pathway were stably integrated into the chromosome of <I>E. coli</I>. The expression levels of chromosomal integrated recombinant biosynthetic genes were varied and adjusted to improve the ratios of carotenoids produced by this <I>E. coli </I>strain. The strategy presented, which combines chromosomal integration of biosynthetic genes with the possibility of adjusting expression by using different promoters, might be useful as a general approach for the construction of stable heterologous production strains synthesizing natural products. This is the case especially for heterologous pathways where excessive protein overexpression is a hindrance.</P>