Optimization Of S-adenosyl-L-methionine Production In Recombinant Pichia Pastoris By Unmarked Chromosomal Manipulation

Unmarked chromosomal manipulation can be used in genetic engineering to realize the recycling of selection markers, thus permitting sequential genetic manipulations in a single host cell. In order to enhance S-adenosyl-L-methionine (SAM) accumulation in the recombinant Pichia pastoris, the unmarked chromosomal manipulation was established and used to further improve SAM synthesis in SAM producing strain DS16which overexpressed a recombinant SAM synthetase gene.A MazF-ZeoR cassette (CYCTT-MazF-ZeoR-CYCTT) was constructed for unmarked chromosomal manipulation, using the combination of an actives-electable (Zeocin resistance, ZeoR) and a counter-selectable (MazF) marker flanked by two direct repeats for marker recycling. Linearized delivery vectors containing MazF-ZeoR cassette were integrated into the P. pastoris genome via homologous recombination. When induced with methanol, the expression of MazF driven by AOX1promoter halted cell growth. Upon counter-selection with methanol, another homologous recombination between CYCTT repeats led to the excision of the selection cassette, thereby realizing the desired alteration to the chromosome without introducing any unwanted selection markers.The weaker promoter G12was used to replace the promoter of Cystathionine-β-synthase (CBS) involved in SAM conversion to down-regulate the expression of CBS, generating the recombinant strain G/CG. In comparison with the original strain DS16, cell growth and MAT activity were not affected in G/CG cultivated in shake flasks, while SAM production was increased by54%.To promote oxygen transmission efficiency and ATP synthesis, Vitreoscilla hemoglobin gene vgb was integrated into the genomic locus of the spe2gene that encodes S-adenosyl-L-methionine decarboxylase involved in SAM conversion. The vgb gene was placed under the control of AOX1or PsADH2promoter to obtain the recombinant strain DS16/DsvA or DS16/DsvP, respectively. The combination of spe2inactivation and vgb overexpression resulted in the increase of not only cell growth by11.8%and23.1%, but also SAM production by17.7%and37.3%, in DS16/DsvP and DS16/DsvA respectively, compared to the original strain DS16.