Construction Of Engineering Strain Pseudomonas Sp. B3-1 And Optimization Of Its Fermentation Conditions For Catechol Production

Catechol(1,2-benzenediol)is an important chemical intermediate for the synthesis of antioxidant,tan-reagent,spice,dyes,photographic material,latex, pesticide and medicine,etc.Up to now,The lower yield of catechol from chemosynthesis and extracted from the natural plants can not meet the fast growing market.With the robust market demand of catechol in our country, more attention has been payed to the new ways to produce catechol. Biosynthesis of catechol by micro-organisms is one of the research hotspots in the world at present.The main work of this paper is to construct engineering bacteria for catechol production and then optimize its culture conditions to increase catechol yield.A complete gene cluster PSB-AD for catechol synthesis from the wild-type strain Pseudomonasp sp.B-1 was cloned using PCR method. Sequence analysis showed that the gene cluster include four intact ORFs,PSB-A, PSB-B,PSB-C and PSB-D.The protein encoded by PSB-AC belonged to the ring hydroxylating dioxygenases which were multicomponent 1,2-dioxygenase complexes.PSB-AB encoded the hydroxylase component which composed of two subunits:alpha-subunit and beta-subunit.PSB-C encoded electron transfer component.The protein encoded by PSB-AC converted closed-ring structures to non-aromatic cis-diols or cis-1,2-Dihydroxybenzoic acid.The peptide encoded by PSB-D was a short-chain dehydrogenase which converted cis-1,2-Dihydroxybenzoic acid to catechol.The versatile broad-host range vectors pK18mob,pLAFRJ and pCM80 harboring the PSB-AC or PSB-AD gene cluster were independently transformed into E.coli DH5αto construct six recombinant strains.The wild strain Pseudomonas sp.B-1 was used as recipient strain for triparental mating with the helper plasmid pRK2073.Conjugants were screened by antibiotics selection, PCR and southern hybridization analysis.As a result,one recombinant strain Pseudomonasp sp.B3-1 showed 10%of catechol yield higher than that of the wild-type strain Pseudomonas sp.B-1 was achieved.To improve its catechlol production,shake flask fermentation were carried out using the engineering strain Pseudomonasp sp.B3-1.The optimized conditions for catechlol accumulation were:sodium benzoate 6.0 g/L, polypepton 2.5 g/L,pH6.0,incubated at 32℃and 200 rpm for 36 hours.The catechol yield improved about 20%compared with that before optimization and reached 0.7 mg/ml.