Recent advances in systems and synthetic biology approaches for developing novel cell-factories in non-conventional yeasts.

S-adenosyl-l-methionine (SAM) is a highly valued chemical that can be used as a dietary supplement and has been used to treat depression, osteoarthritis, and liver problems as well. We adopted systems metabolic engineering strategies to improve SAM production in a high-producing strain (GS115/DS56). First, the cystathionine β-synthase gene CYS4 was downregulated using a weak promoter PG12 to reduce the removal of homocysteine from SAM cycle, thus leading to a 48.8% increase in the SAM titer (1.68 g/L) from the strain G12-CBS, while preventing cysteine auxotrophy induced by deletion of this essential gene. Subsequently, the SAM titer of G12-CBS was improved to 13.01 g/L in 15-L fed-batch fermentation using the optimal l-methionine feeding strategy. Finally, based on comparative transcriptomics, five genes were chosen and overexpressed for further enhancement of SAM production. Among them, GDH2 and ACS2 exhibited positive effects, and the additional overexpression of GDH2 led to a 52.3% increase of titer (2.71 g/L) in shake flask culture. Therefore, the engineered Pichia pastoris strains can be utilized in industrial production of SAM using a simple and cost-effective process, and these approaches could be employed for improving the production of other chemicals by P. pastoris.

Engineering Pichia pastoris to improve S-adenosyl- l-methionine production using systems metabolic strategies.

Predominance of indigenous non-Saccharomyces yeasts in the traditional fermentation of greengage wine and their significant contribution to the evolution of terpenes and ethyl esters.

Two genes encoding methionine adenosyltransferase, SAM2 from Saccharomyces cerevisiae and metK from Corynebacterium glutamicum, were individually cloned into pDXW-8, the shuttle vector between Escherichia coli and C. glutamicum, and overexpressed in E. coli DH5α and C. glutamicum ATCC13032. In DH5α, both genes were overexpressed and their protein products showed the activity of methionine adenosyltransferase. In ATCC13032, metK was overexpressed, its product MetK showed the enzyme activity and could convert l-methionine to S-adenosyl-l-methionine (SAM). However, when SAM2 was overexpressed in ATCC13032, neither the enzyme activity nor the conversion of SAM from l-methionine was observed. Reverse transcription PCR analysis and SDS-PAGE showed that SAM2 was transcribed but not translated in C. glutamicum. Therefore, SAM2-C, a mutant SAM2, was constructed by codon optimization, and overexpressed in ATCC13032; it was well transcribed and translated, and could convert l-methionine to SAM. Finally, SAM2-C and metK were individually overexpressed in E. coli BL21(DE3), and their products SAM2-C and MetK were purified and characterized. The optimum activity for both enzymes was found at pH 8.5 and 35 °C; SAM2-C and MetK have similar Km for ATP, but quite different Km for l-methionine. These results suggest that SAM2-C and MetK can be useful for developing C. glutamicum to produce SAM.

Overexpression of methionine adenosyltransferase in Corynebacterium glutamicum for production of S-adenosyl-l-methionine

Abstract Background The methylotrophic budding yeast Pichia pastoris GS115 is a powerful expression system and hundreds of heterologous proteins have been successfully expressed in this strain. Recently, P. pastoris has also been exploited as an attractive cell factory for the production of high-value biochemicals due to Generally Recognized as Safe (GRAS) status and high growth rate of this yeast strain. However, appropriate regulation of metabolic flux distribution between cell growth and product biosynthesis is still a cumbersome task for achieving efficient biochemical production. Results In this study, P. pastoris was exploited for high inositol production using an effective dynamic regulation strategy. Through enhancing native inositol biosynthesis pathway, knocking out inositol transporters, and slowing down carbon flux of glycolysis, an inositol-producing mutant was successfully developed and low inositol production of 0.71 g/L was obtained. The inositol production was further improved by 12.7% through introduction of heterologous inositol-3-phosphate synthase (IPS) and inositol monophosphatase (IMP) which catalyzed the rate-limiting steps for inositol biosynthesis. To control metabolic flux distribution between cell growth and inositol production, the promoters of glucose-6-phosphate dehydrogenase (ZWF), glucose-6-phosphate isomerase (PGI) and 6-phosphofructokinase (PFK1) genes were replaced with a glycerol inducible promoter. Consequently, the mutant strain could be switched from growth mode to production mode by supplementing glycerol and glucose sequentially, leading to an increase of about 4.9-fold in inositol formation. Ultimately, the dissolved oxygen condition in high-cell-density fermentation was optimized, resulting in a high production of 30.71 g/L inositol (~ 40-fold higher than the baseline strain). Conclusions The GRAS P. pastoris was engineered as an efficient inositol producer for the first time. Dynamic regulation of cell growth and inositol production was achieved via substrate-dependent modulation of glycolysis and pentose phosphate pathways and the highest inositol titer reported to date by a yeast cell factory was obtained. Results from this study provide valuable guidance for engineering of P. pastoris for the production of other high-value bioproducts. 

https://microbialcellfactories.biomedcentral.com/counter/pdf/10.1186/s12934-022-01837-x

Metabolic engineering of Pichia pastoris for myo-inositol production by dynamic regulation of central metabolism

Methionine adenosyltransferase (MAT, EC2.5.1.6) catalyzes the synthesis of S-adenosylmethionine (SAM) using l-methionine and adenosine triphosphate (ATP) as substrates. The mutant MAT pDS16 was obtained through DNA shuffling previously in our lab. Overexpression of pDS16 in Pichia pastoris led to about 65 % increase of MAT activity and SAM accumulation, compared with the strain overexpressing Saccharomyces cerevisiae MAT gene SAM2. Different strategies were tested to facilitate the expression and purification of pDS16. However, addition of the hexahistidine tag to pDS16 was shown to decrease the enzyme activity, and the yeast α-factor signal sequence could not effectivley direct the secretion of pDS16. The intracellular pDS16 was purified by a simple two-step procedure combining an ion exchange and hydrophobic interaction chromatography. Protein purity was verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis to be 93 %, with the specific activity of 1.828 U/mg. Two-dimensional electrophoresis revealed pI of ∼5.5. The purified enzyme followed Michaelis kinetics with a Km of 1.72 and 0.85 mM, and Vmax of 1.54 and 1.15 μmol/min/mg for ATP and L-methionine, respectively. pDS16 exhibited optimal activity at pH 8.5 and 45 °C with the requirement of divalent cation Mg2+ and was slightly stimulated by the monovalent cation K+. It showed an improved thermostability, about 50 % of the enzyme activity was retained even after preincubation at 50 °C for 2 h.

Expression, purification, and characterization of a recombinant methionine adenosyltransferase pDS16 in Pichia pastoris.

The invention relates to a method for enhancing yield of S-adenosylmethionine by regulating gln1 gene expression The coding gene of glutamine synthetase is transformed into S-adenosylmethionine to produce the strain for the first time, thereby obviously enhancing the yield of S-adenosylmethionine of the S-adenosylmethionine production strain

Method for enhancing yield of S-adenosylmethionine by regulating gln1 gene expression

The invention relates to an application of a GAP (GTPase-Activating Protein) promoter library in the regulation of a metabolic pathway of S-adenosylmethionine and also provides an expression vector for regulating a metabolic pathway of S-adenosylmethionine and a host cell containing the expression vector. According to the invention, a constitutive promoter library is adopted so as to realize the precise regulation on the gene expression in the metabolic pathway of the S-adenosylmethionine, and a feasible method for increasing the yield of the S-adenosylmethionine is found.

Xiaole Wu

Papers

Engineering Pichia pastoris to improve S-adenosyl- l-methionine production using systems metabolic strategies.

Expression, purification, and characterization of a recombinant methionine adenosyltransferase pDS16 in Pichia pastoris.

Method for enhancing yield of S-adenosylmethionine by regulating gln1 gene expression

Application of GAP (GTPase-Activating Protein) promoter library in regulation of metabolic pathway of S-adenosylmethionine