Carotenoids are pigments that are ubiquitous throughout nature and synthesized by all photosynthetic organisms and in some heterotrophic bacteria and fungi. Carotenoids provide color for flowers, vegetables, insects, fish and birds. Colors of carotenoid range from yellow to red with variations of brown and purple. As precursors of vitamin A, carotenoids are fundamental components in the human diet and play an important role in human health. Animals are unable to synthesize carotenoids de novo and must obtain them through diet. Manipulation of carotenoid composition and production in plants or bacteria can provide new and/or improved sources of carotenoids. Industrial uses of carotenoids include, among others, pharmaceuticals, food supplements, animal feed additives, and colorants in cosmetics.
The genetics of carotenoid biosynthesis are well known (Armstrong, G., in Comprehensive Natural Products Chemistry, Elsevier Press, volume 2, pp 321–352 (1999)); Lee, P. and Schmidt-Dannert, C., Appl Microbiol Biotechnol, 60:1–11 (2002); Lee et al., Chem Biol 10:453–462 (2003), and Fraser, P. and Bramley, P. (Progress in Lipid Research, 43:228–265 (2004)). This pathway is extremely well studied in the Gram-negative, pigmented bacteria of the genera Pantoea, formerly known as Erwinia. Of particular interest are the genes responsible for the production of C40 carotenoids used as pigments in animal feed (e.g., canthaxanthin and astaxanthin).
The genes associated with carotenoid biosynthesis (C40) can be generally divided into two categories of genes: 1) the C40 carotenoid backbone biosynthesis genes responsible for the elongation, desaturation, and cyclization steps necessary for the synthesis of the 40-carbon backbone (i.e., the crtE, crtB, crtI, and crtY genes responsible for the biosynthesis of β-carotene) and 2) subsequent carotenoid modification genes (i.e., crtW, crtO, crtZ, etc.), which introduce various functional groups (e.g., keto groups and hydroxyl groups) to the 40-carbon backbone. The biosynthesis of ketocarotenoids and hydroxylated carotenoids is of particular interest as they are commercially important pigments (e.g., canthaxanthin, astaxanthin, zeaxanthin, etc.) used in a variety of applications, including the animal feed market. Recombinant expression of the genes involved in carotenoid production has been reported in a variety of hosts.
Ketocarotenoid biosynthesis typically requires expression of a carotenoid ketolase. Two classes of carotenoid ketolase have been reported (CrtW/bkt and CrtO). The two classes have similar functionality yet appear to have arisen independently as they share very little sequence similarity (U.S. Pat. No. 6,984,523 and U.S. Ser. No. 11/015,433, each incorporated herein by reference). Carotenoid ketolases introduce keto groups to the ionone ring of cyclic carotenoids forming ketocarotenoids including, but not limited to echinenone, 3-hydroxyechinenone, 3′-hydroxyechinenone, adonixanthin, adonirubin, canthaxanthin and astaxanthin.
Biosynthesis of hydroxylated carotenoids typically requires expression of a carotenoid hydroxylase. Carotenoid hydroxylases introduce hydroxyl groups to the ionone ring of the cyclic carotenoids, such as β-carotene or canthaxanthin. Bacterial biosynthesis of astaxanthin requires functional expression of both a carotenoid ketolase and a CrtZ carotenoid hydroxylase, which is encoded by a crtZ gene as reported in U.S. Ser. No. 11/200,394, incorporated herein by reference. Besides astaxanthin, examples of hydroxylated carotenoids include β-cryptoxanthin, zeaxanthin, 3-hydroxyechinenone, 3′-hydroxyechinenone, adonirubin, adonixanthin, tetrahydroxy-β,β′-caroten-4,4′-dione, tetrahydroxy-β,β′-caroten-4-one, caloxanthin, erythroxanthin, nostoxanthin, flexixanthin, 3-hydroxy-γ-carotene, 3-hydroxy-4-keto-γ-carotene, bacteriorubixanthin, bacteriorubixanthinal, and lutein.
It has been reported for microbial carotenoid production that the concentration of dissolved oxygen within a microbial culture affects the carotenoid production profile (U.S. Pat. No. 6,825,002 to Tsubokura et al.). This is because both carotenoid ketolases and carotenoid hydroxylases require molecular oxygen to synthesize canthaxanthin and astaxanthin. Conversion of β-carotene to canthaxanthin and/or astaxanthin, as well as various intermediates in the pathway, can be adversely affected under oxygen limited conditions.
A problem in large-scale fermentation is that increasing dissolved oxygen mechanistically is costly and often not workable. A biological method is therefore needed to increase the overall availability of cellular oxygen, that is, to increase internal O2 tension within a recombinant microbial cell. One way to do this is to increase the cellular components that aid in the intracellular storage and delivery of oxygen. It has been reported that bacterial hemoglobins, a subset of the larger hemoglobin-like superfamily, perform these functions (Frey, A. D. and Kallio, P. T., FEMS Microbiol Rev. 27:525–545 (2003)). Three different types of bacterial hemoglobins have been identified: 1) the Vitreoscilla hemoglobin (VHb), 2) the flavohemoglobins (FHb), and 3) the truncated hemoglobins (trHb). The truncated hemoglobins are further divided into 3 groups, Group I (HbN-type), Group II (HbO-type), and Group III (HbP-type). All bacterial hemoglobins are able to reversibly bind molecular oxygen.
The Vitreoscilla hemoglobin (VHb) is the most widely studied bacterial hemoglobin. Recombinant expression of VHb has been reported to improve the growth characteristics and productivity of various proteins in microorganisms grown under microaerobic/oxygen limited conditions (Frey, A. D. and Kallio, P. T., supra; Bollinger et al., Biotechnol. Prog. 17:798–808 (2001); and U.S. Pat. No. 5,049,493 to Khosla et al.).
However, a method of using recombinant bacterial hemoglobin expression to alter carotenoid titer and/or production of oxygenated carotenoids (i.e., xanthophylls such as canthaxanthin and/or astaxanthin) has not been reported. Furthermore, a method of using a truncated bacterial hemoglobin, which is structurally unrelated to the Vitreoscilla hemoglobin, to improve overall growth and/or carotenoid production in a recombinant microbial host cell has not been reported.
Recombinant expression of truncated bacterial hemoglobins from Mycobacterium tuberculosis has been reported (Pathania et al., J. Biol. Chem., 277:15293–15302 (2002)). Pathania et al. report that the truncated hemoglobins HbN and HbO from M. tuberculosis share little structural similarity in their EF-loop regions, suggesting distinct function(s) for each. Recombinant expression of the M. tuberculosis HbO resulted in a significant increase in cell mass and higher oxygen update in aerobically growing cells. Given the desirable effects on overall cell growth, there remains a need to identify additional truncated bacterial hemoglobins, especially from non-pathogenic organisms, useful for industrial biotechnology.
The problem to be solved therefore is to provide an isolated nucleic acid molecule encoding a truncated bacterial hemoglobin isolated from a non-pathogenic microorganism capable of increasing the growth rate and/or carotenoid production when recombinantly expressed in a carotenogenic host cell.