Optimization Of Gellan Production And Functional Analysis Of Genes Involved In Nostoxanthin Biosynthesis Of Sphingomonas

Sphingomonas elodea ATCC31461was isolated as a Gram-negative bacterium capable of producing gellan gum. Gellan has been used as a suspending, gelling, stabilizing, thickening, emulsifying, texturing agent in the food, pharmaceutical, chemical and other industries. Moreover, its potential use as a replacement for gelatin and agar makes it the most important commercialized bacterial exopolysaccharide, and market demand for it has been increasing. However, its relatively low productivity, high stirring energy consumption and costly downstream purification process limit its economic viability. Most Sphingomonas synthesize a yellow carotenoid identified as nostoxanthin. However, the genes responsible for nostoxanthin synthesis have not been identified from any Sphingomonas species so far. During the synthesis of gellan, Sphingomonas elodea ATCC31461also accumulates yellow carotenoid pigments, mainly nostoxanthin. Furthermore, the viscosity of broth dramatically increases throughout the fermentation process and reaches a high value at the end of the process. These characteristics not only affect fermentation efficiency but also make it difficult to separate gellan from the fermentation broth. In the general procedure for recovering gellan, large amounts of isopropanol or ethanol have been used for precipitating the biopolymer, as well as removing pigments and other impurities, thereby increasing production costs. This study is aimed to optimize gellan production (including strain improvent and develop a low-cost method for gellan recovery) as well as cloning and characterization of genes involved in nostoxanthin biosynthesis of Sphingomonas elodea ATCC31461. The main results are as follows:1. Strain improvement by mutation. A carotenoid-free mutant strain of ATCC31461(βm007) was isolated by ethyl methanesulfonate mutagenesis combined with ultraviolet irradiation. The final gellan production of the mutant strain was13%higher than that of the parent strain. The glycerate and acetate substituents of gellan produced by the mutant strain were17%and65%higher than that produced from ATCC31461, respectively. Stability tests suggested that the broth produced by the mutant strain was more stable during storage at room temperature compared with that produced by the parent strain. The mutant strain seems to be an ideal strain for use in the commercial production of gellan.2. Cloning and knockout of phytoene desaturase gene. The phytoene desaturase gene(crtl) in the carotenoid bio synthetic pathway of S. elodea ATCC31461was cloned and identified. This gene contained1479bp and encoded a492-amino acid protein with significant homo logy to the phytoene desaturase of other carotenogenic organisms. Knockout of crtl gene blocked yellow carotenoid pigment synthesis and resulted in the accumulation of colorless phytoene, confirming that it encodes phytoene desaturase. Further research indicates that the yield of gellan gum production and cell growth by crtl gene knockout mutants (△crtl) is almost the same as that by the wild-type strain. Consequently, the△crtl mutant can be used as an industrial gellan gum-producing strain in place of ATCC31461.3. Develop a low-cost method for gellan recovery. Based on the non-pigment fermentation broths of βm007and the△crtl mutant, we designed and analyzed an economical recovery method. In this method, a200g/L CaCl2solution was used to flocculate gellan, followed by precipitation with30%ethanol (v/v). The recovery yield of gellan (including the unavoidable impurities) obtained from△crtl through this new method was quite close to that from ATCC31461recovered via the conventional method. Compared to the volume of alcohol for the parent strain (2volumes of broth), much less alcohol (30%) is required in the new recovery process; thus, the costs of downstream purification of gellan gum can be substantially reduced.4. Cloning and characterization of genes involved in nostoxanthin biosynthesis. A carotenoid biosynthesis gene cluster containing four carotenogenic genes (crtG, crtY, crtl and crtB) and a β-carotene hydroxylase gene (crtZ) located outside the cluster were cloned and characterized. The individual ATCC31461carotenogenic gene products showed moderate identity to the corresponding proteins derived from Brevundimonas (also synthesize nostoxanthin), ranging from43%to58%. Each of these genes was inactivated, and the biochemical function of each gene was confirmed based on chromatographic and spectroscopic analysis of the intermediates accumulated in the knockout mutants. Moreover, the crtG gene encoding the2,2’-β-hydroxylase and the crtZ gene encoding the β-carotene hydroxylase, both responsible for hydroxylation of P-carotene, were confirmed by complementation studies using Escherichia coli producing different carotenoids. Expression of crtG in zeaxanthin and p-carotene carotene accumulating E. coli cells resulted in the formation of nostoxanthin and2,2’-dihydroxy-β-carotene, respectively. Based on these results, a biochemical pathway for synthesis of nostoxanthin in S. elodea ATCC31461is proposed.