ABSTRACT
BACKGROUND: In synthetic biology, the strength of promoter elements is the basis for precise regulation of target gene transcription levels, which in turn increases the yield of the target product. However, the results of many researches proved that excessive transcription levels of target genes actually reduced the yield of the target product. This phenomenon has been found in studies using different microorganisms as chassis cells, thus, it becomes a bottleneck problem to improve the yield of the target product. RESULTS: In this study, promoters PGK1p and TDH3p with different strengths were used to regulate the transcription level of alcohol acetyl transferase encoding gene ATF1. The results demonstrated that the strong promoter TDH3p decreased the production of ethyl acetate. The results of Real-time PCR proved that the transcription level of ATF1 decreased rapidly under the control of TDH3p, and the unfolded protein reaction was activated, which may be the reason for the abnormal production caused by the strong promoter. RNA-sequencing analysis showed that the overexpression of differential gene HSP30 increased the transcriptional abundance of ATF1 gene and production of ethyl acetate. Interestingly, deletion of the heat shock protein family (e.g., Hsp26, Hsp78, Hsp82) decreased the production of ethyl acetate, suggesting that the Hsp family was also involved in the regulation of ATF1 gene transcription. Furthermore, the results proved that the Hsf1, an upstream transcription factor of Hsps, had a positive effect on alleviating the unfolded protein response and that overexpression of Hsf1 reprogramed the pattern of ATF1 gene transcript levels. The combined overexpression of Hsf1 and Hsps further increased the production of ethyl acetate. In addition, kinase Rim15 may be involved in this regulatory pathway. Finally, the regulation effect of Hsf1 on recombinant strains constructed by other promoters was verified, which confirmed the universality of the strategy. CONCLUSIONS: Our results elucidated the mechanism by which Rim15-Hsf1-Hsps pathway reconstructed the repression of high transcription level stress and increased the production of target products, thereby providing new insights and application strategies for the construction of recombinant strains in synthetic biology.
ABSTRACT
Baijiu is a traditional distilled beverage in China with a rich variety of aroma substances. 2,3,5,6-tetramethylpyrazine (TTMP) is an important component in Baijiu and has the function of promoting cardiovascular and cerebrovascular health. During the brewing of Baijiu, the microorganisms in jiuqu produce acetoin and then synthesize TTMP, but the yield of TTMP is very low. In this work, 2,3-butanediol dehydrogenase (BDH) coding gene BDH1 and another BDH2 gene were deleted or overexpressed to evaluate the effect on the content of acetoin and TTMP in Saccharomyces cerevisiae. The results showed that the acetoin synthesis of strain α5-D1B2 was significantly enhanced by disrupting BDH1 and overexpressing BDH2, leading to a 2.6-fold increase of TTMP production up to 10.55 mg/L. To further improve the production level of TTMP, the α-acetolactate synthase (ALS) of the pyruvate decomposition pathway was overexpressed to enhance the synthesis of diacetyl. However, replacing the promoter of the ILV2 gene with a strong promoter (PGK1p) to increase the expression level of the ILV2 gene did not result in further increased diacetyl, acetoin and TTMP production. Based on these evidences, we constructed the diploid strains AY-SB1 (ΔBDH1:loxP/ΔBDH1:loxP) and AY-SD1B2 (ΔBDH1:loxP-PGK1p-BDH2-PGK1t/ΔBDH1:loxP-PGK1p-BDH2-PGK1t) to ensure the fermentation performance of the strain is more stable in Baijiu brewing. The concentration of TTMP in AY-SB1 and AY-SD1B2 was 7.58 and 9.47 mg/L, respectively, which represented a 2.3- and 2.87-fold increase compared to the parental strain. This work provides an example for increasing TTMP production in S. cerevisiae by genetic engineering, and highlight a novel method to improve the quality and beneficial health attributes of Baijiu.
ABSTRACT
Appropriate concentrations and proportion of acetate esters and higher alcohols improve the quality of Chinese Baijiu. To regulate the concentrations of acetate esters in Chinese Baijiu, we constructed a PGK1 promoter library through error-prone PCR. Then, we used an enhanced green fluorescent protein as a reporter to characterize the activities of PGK1p mutants. The PGK1p library contained 28 PGK1p mutants and spanned an activity that ranged between 0.1% and 141% of wild-type PGK1p. Seven PGK1p mutants were characterized by an additional reporter ß-galactosidase and then used for the overexpression of ATF1 with BAT2 deletion in Saccharomyces cerevisiae a45. The production of ethyl acetate in strains A8, A17, A18, A27, A22, A25, A28, and AWT were 1.66-, 3.09-, 10.59-, 13.07-, 15.99-, 22.67-, 24.06-, and 27.22-fold higher than that of the parental strain. The results on alcohol acetyltransferase (AATase) activity showed that the PGK1p library precisely controlled ATF1 expression and regulated the acetate esters production.
