Construction Of Functional GAP Promoter Library Pave The Way For Metabolic Engineering Of S-Adenosylmethionine Production In Pichia Pastoris

S-Adenosylmethionine (SAM), an important molecule for normal cell function and survival, is synthesized from ATP and L-methionine (L-Met) catalyzed by methionine adenosyltransferase (MAT). Considerable interest in SAM production has arisen because it can effectively treat affective disorders, liver disease, and neurological disorders. In our previous research, overexpression of heterologous MAT gene (ds16) driven by AOX1 promoter has been carried out to enhance SAM accumulation in Pichia pastoris. However, it was found that the degree of increased MAT activity was much higher than that of SAM production. This result implied that MAT activity was not the rate-limiting factor for SAM synthesis in this recombinant strain (DS16). For further improving SAM production, GAP promoter (PGAP) library was constructed and applied for precise transcriptional control of dsl6 and cystathionine-βsynthase (CBS) gene cys4 in DS16, which determine MAT activity for SAM synthesis and the SAM transsulfuration pathway flux.To develop a reliable and sensitive high-throughput protocol screening for the engineered constitutive promoters in the yeast P. pastoris, the yeast-enhanced green fluorescent protein (yEGFP) was used as the reporter to monitor the promoter strength. After eliminating the interfering components (yeast extract and tryptone) with fluorescence signal from the medium, a high-throughput screening approach was established and optimized to obtain a low standard deviation of cell density (6.9%) and fluorescence (7.4%) in 48-deep-well microplates. Then 300 clones containing PGAP variants were screened, exhibiting a wide range in fluorescent intensity from about 8% to 218% of that obtained with PGAP. Six representative clones with unique promoter sequence were picked for further characterization. A good correlation between yEGFP fluorescence in microplates and shake flasks was observed. Furthermore, the high correlation between fluorescence and mRNA level confirmed that expression was transcriptionally controlled. Therefore, a reliable high-throughput screening approach was developed for selection of engineered constitutive promoters with different activites.Nearly 30,000 promoter mutants were screened by using the high-throughput approach. 33 mutants, spanning an activity range between～0.6% and 19.6-fold of the wild-type promoter activity at the level of fluorescence intensity with an almost linear distribution, were finally chosen to form a functional promoter library. After an extensive characterization of the library, the broader applicability of the results obtained with the yEGFP reporter was confirmed using two additional reporters [P-galactosidase and methionine adenosyltransferase (MAT)] at the transcription and enzyme activity levels. Furthermore, the utility of the promoter library was tested by investigating the impact of the heterologous MAT gene expression levels on cell growth and SAM production. The full characterization of the promoter strength enabled identification of the optimal MAT specifity activity (around 1.05 U/mg of protein) to obtain the maximal volumetric SAM production.The further increasing of MAT activity can not enhance SAM production in DS16. Based on this observation, cystathionine-βsynthase (CBS), an enzyme involved in SAM transsulfuration, was expressed under control of different mutants of GAP promoter. To obtain higher SAM production, the SAM transsulfuration pathway through cystathionine was engineered by down regulation of cystathionine-βsynthase (CBS) which catalyzes the synthesis of cystathionine from homocysteine. Therefore, the CBS promoter in DS16 was replaced by three weak promoters (PG0, PG6 and PG12) to construct the corresponding the recombinant strains GO-CBS, G6-CBS and G12-CBS. While no apparent difference in cell growth between recombinant strains (GO-CBS, G6-CBS, G12-CBS) and the control strain DS16 was observed, the analysis of the SAM accumulation revealed that the specific SAM production decreased by 9.2% in GO-CBS but increased 23.8% and 31.2% in G6-CBS and G12-CBS respectively, compared to DS16. In addition, the CBS activity was decreased by 14% and 73% in G6-CBS and G12-CBS respectively, whereas improved by 15% in GO-CBS. Moreover, the similar trend was observed for cys4 gene transcription levels. Therefore, weaker promoters drove CBS expression is efficiently as expected.Fermentation in 5 L and 15 L scale was examined, recombinant strain G12-CBS was cultivated in 5 L bioreactor. Compared with DS16, the SAM accumulation (6.7 g/L) and L-Met conversion rate (34.6%) of G12-CBS were improved by 48.8% and 78.4% respectively. Then, recombinant strain G12-CBS was cultivated in 15 L bioreactor for SAM production. The volumetric production of SAM (11.9 g/L) and L-Met conversion rate (49.5%) of G12-CBS were improved by 1.2- and 1.5-flold respectively, compared with DS16. In conclusion, the genomic integration of the mutated promoter PG12 upstream of cys4 gene, down regulated CBS expression, which markedly enhanced SAM production and L-Met conversion rate in 5- and 15-L bioreactors. In order to enhance SAM production through blocking alternative SAM transformation pathways, S-adenosylmethionine decarboxylase gene spe2, S-adenosyl-L-homocysteine hydrolase gene sahl, and L-methylthio-tRNA synthase gene msm1 was knocked out in DS16 generating the recombinant strain G/Dspe, G/Dmsm and G/Dsah, respectively. Cell growth and SAM synthesis of the three knockout strains were investigated. The knock-out had no effect on cell growth, but, SAM production was improved by 29.3%,55.6%, and 24.8% in G/Dspe, G/Dsah, and G/Dmsm, respectively, compared to the original strain DS16. Additionally, the effect of methionine addition on SAM accumulation was investigated. When methionine addition was decreased from 0.1% to 0.06%, SAM production was increased by 7.1% and 9.5% in G/Dsah and G/Dmsm respectively.