| Summary: | <p>Abstract</p> <p>Background</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 <it>Haematococcus pluvialis</it>, heterologous biosynthesis in non-carotenogenic microorganisms like <it>Escherichia coli</it>, is a promising alternative for sustainable production of natural astaxanthin. Recent achievements in the metabolic engineering of <it>E. coli </it>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 <it>E. coli</it>, however, the conversion of β-carotene to astaxanthin is obviously the most critical step towards an efficient biosynthesis of astaxanthin.</p> <p>Results</p> <p>Here we report the construction of the first plasmid-free <it>E. coli </it>strain that produces astaxanthin as the sole carotenoid compound with a yield of 1.4 mg/g cdw (<it>E. coli </it>BW-ASTA). This engineered <it>E. coli </it>strain harbors xanthophyll biosynthetic genes from <it>Pantoea ananatis </it>and <it>Nostoc punctiforme </it>as individual expression cassettes on the chromosome and is based on a β-carotene-producing strain (<it>E. coli </it>BW-CARO) recently developed in our lab. <it>E. coli </it>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 <it>crtEBIY </it>along with the β-carotene-ketolase gene <it>crtW148 </it>(NpF4798) and the β-carotene-hydroxylase gene (<it>crtZ</it>) under controlled expression conditions in <it>E. coli </it>BW-ASTA directed the pathway exclusively towards the desired product astaxanthin (1.4 mg/g cdw).</p> <p>Conclusions</p> <p>By using the λ-Red recombineering technique, genes encoding for the astaxanthin biosynthesis pathway were stably integrated into the chromosome of <it>E. coli</it>. The expression levels of chromosomal integrated recombinant biosynthetic genes were varied and adjusted to improve the ratios of carotenoids produced by this <it>E. coli </it>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>
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