Producing malonate in Saccharomyces cerevisiae via theβ‑alanine pathway

Malonate is a high-value chemical that can be used to produce value-added compounds.Due to the toxic by-products and low product yield for malonate production through hydrolysis of cyanoacetic acid,microbial production methods have attracted significant attention.Previously,theβ-alanine pathway has been engineered in Escherichia coli for malonate production.In this study,theβ-alanine pathway was constructed in Saccharomyces cerevisiae by introducing the heterologous genes of BcBAPAT and TcPAND to convert l-aspartate to malonic semialdehyde,combining with co-expression genes of AAT2 and UGA2 to improve precursor supply and malonate producing.Through delta sequence-based integration of the two heterologous genes,the engineered strain produced with 7.21 mg/L malonate was screened.Further,replaced the succinic semialdehyde dehydrogenase gene UGA2 with yneI from E.coli which was utilized to produce malonate in previous study,increased the malonate titer to 7.96 mg/L in flask culture.Following optimization,fermentation of the final engineered strain in shake flasks yielded a maximum malonate titer of 12.83 mg/L,and this was increased to 91.53 mg/L during fed-batch fermentation in a 5 L bioreactor which increased by two-fold compared with that of the engineered strain overexpressing UGA2.

Identification and development of a glucaric acid biosensor in Saccharomyces cerevisiae

Metabolic biosensors are increasingly used in metabolic engineering and synthetic biology.In this study,using Saccha-romyces cerevisiae as a model system,we developed a methodology to identify promoter elements that are responsive to glucaric acid.Through transcriptome analysis,it was found that multiple genes were upregulated when cells were exposed to high concentrations of glucaric acid.From the promoters of these candidate genes,the YCR012W promoter(PYCR012W)was observed to specifically respond to glucaric acid in a dose-dependent manner.To gain further insight into the binding site of glucaric acid-responsive activators,we truncated the promoter and revealed that the-564 to-464 bp regions of PYCR012W was essential for glucaric acid-responsive expression.To investigate the glucaric acid-responsive transcription factors,we predicted the transcription factor binding sites in the-564 to-464 bp region of PYCR012W and found that two transcription factors,Ash1p and Cbf1p,might be linked to glucaric acid responses.The strategies used in this study outline a method for the identification and development of metabolic biosensors.