In recent years, many studies have been conducted on the expression of multiple aromatic compounds by Saccharomyces cerevisiae. The concentration of L-tyrosine, as a precursor of such valuable compounds, is crucial for the biosynthesis of aromatic metabolites. In this study, a novel function of HTZ1 was found to be related to tyrosine biosynthesis, which has not yet been reported. Knockout of this gene could significantly improve the ability of yeast cells to synthesize tyrosine, and its p-coumaric acid (p-CA) titer was approximately 3.9-fold higher than that of the wild-type strain BY4742. Subsequently, this strain was selected for random mutagenesis through an emerging mutagenesis technique, namely, atmospheric and room temperature plasma (ARTP). After two rounds of mutagenesis, five tyrosine high-producing mutants were obtained. The highest production of p-CA was 7.6-fold higher than that of the wild-type strain. Finally, transcriptome data of the htz1Δ strain and the five mutants were analyzed. The genome of mutagenic strains was also resequenced to reveal the mechanism underlying the high titer of tyrosine. This system of target engineering combined with random mutagenesis to screen excellent mutants provides a new basis for synthetic biology.

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Aims To overcome the defective unstable production of p‐coumaric acid (p‐CA) using episomal plasmids and simultaneously achieve genetic stability and high‐copy integration in Saccharomyces cerevisiae. Methods and results Two‐micron plasmids were used to obtain high titres of p‐CA, but p‐CA production was decreased significantly in a nonselective medium after 72 h. To overcome the defect of unstable p‐CA production during fermentation, delta integration with the triosephosphate isomerase gene from Schizosaccharomyces pombe (POT1) was employed as a selection marker to integrate heterologous p‐CA synthesis cassette, and the high‐level p‐CA‐producing strain QT3‐20 was identified. In shake flask fermentation, the final p‐CA titre of QT3‐20 reached 228.37 mg L−1 at 168 h, 11‐fold higher than integrated strain QU3‐20 using URA3 as the selective marker, and 9‐fold higher than the best‐performing episomal expression strain NKE1. Additionally, the p‐CA titre and gene copy number remained stable after 100 generations of QT3‐20 in a nonselective medium. Conclusion We achieved high‐copy genome integration and stable heterologous production of p‐CA via a POT1‐mediated strategy in S. cerevisiae. Significance and impact of study With superior genetic stability and production stability in a nonselective medium during fermentation, the high‐level p‐CA‐producing strain constructed via POT1‐mediated delta integration could serve as an efficient platform strain, to eliminate the threat of unstable and insufficient supply for future production of p‐CA derivatives, make downstream processing and biosynthesis much simpler.

Caffeic acid, a plant-sourced phenolic compound, has a variety of biological activities, such as antioxidant and antimicrobial properties. The caffeic acid biosynthetic pathway was initially constructed in S. cerevisiae, using codon-optimized TAL (coTAL, encoding tyrosine ammonia lyase) from Rhodobacter capsulatus, coC3H (encoding p-coumaric acid 3-hydroxylase) and coCPR1 (encoding cytochrome P450 reductase 1) from Arabidopsis thaliana in 2 μ multi-copy plasmids to produce caffeic acid from glucose. Then, integrated expression of coTAL via delta integration with the POT1 gene (encoding triose phosphate isomerase) as selection marker and episomal expression of coC3H, coCPR1 using the episomal plasmid pLC-c3 were combined, and caffeic acid production was proved to be improved. Next, the delta and rDNA multi-copy integration methods were applied to integrate the genes coC3H and coCPR1 into the chromosome of high p-coumaric acid yielding strain QT3-20. The strain D9 constructed via delta integration outperformed the other strains, leading to 50-fold increased caffeic acid production in optimized rich media compared with the initial construct. The intercomparison between three alternative multi-copy strategies for de novo synthesis of caffeic acid in S. cerevisiae suggested that delta-integration was effective in improving caffeic acid productivity, providing a promising strategy for the production of valuable bio-based chemicals in recombinant S. cerevisiae.