Douchi is a traditional Chinese fermented soybean product, in which acetoin is a key flavor substance. Here, the α-acetolactate decarboxylase gene aldC was cloned from Lactiplantibacillus (L.) plantarum and overexpressed in Lactococcus (L.) lactis NZ9000 by nisin induction. The ALDC crude enzyme solution produced an enzyme activity of 35.16 mU. Next, whole cells of the recombinant strain NZ9000/pNZ8048-aldC were employed as the catalyst to produce acetoin in GM17 medium. An optimization experiment showed that an initial OD600 of 0.6, initial pH of 7.5, nisin concentration of 20 ng/mL, induction temperature of 37 °C and static induction for 8 h were the optimal induction conditions, generating the maximum acetoin production (106.93 mg/L). Finally, after incubation under the optimal induction conditions, NZ9000/pNZ8048-aldC was used for whole-cell biocatalytic acetoin production, using soybean as the substrate. The maximum acetoin yield was 79.43 mg/L. To our knowledge, this is the first study in which the aldC gene is overexpressed in L. lactis and whole cells of the recombinant L. lactis are used as a biocatalyst to produce acetoin in soybean. Thus, our study provides a theoretical basis for the preparation of fermented foods containing high levels of acetoin and the biosynthesis of acetoin in food materials.

BackgroundTryptophol (TOL) is a metabolic derivative of tryptophan (Trp) and shows pleiotropic effects in humans, plants and microbes. The mechanisms of TOL biosynthesis were first explored several decades ago. Nonetheless, a systematic interpretation of TOL over-accumulation is still lacking.ResultsBased on TOL yield, a suitable transformation medium (TM1) was used to culture Saccharomyces cerevisiae strain KMLY1-2. The dynamics of TOL production, cell growth, and gene transcription revealed that TOL production was dependent on cell density and the expression of key genes. Additionally, the effects of Trp and phenylalanine (Phe) on TOL production were tested, and the results showed that Trp can significantly facilitate TOL accumulation, but output plateaued (231.02−266.31 mg/L) at Trp concentrations ≥0.6 g/L. In contrast, Phe reduced the stimulatory effect of Trp, which strongly depended on the Phe concentration. To elucidate the molecular basis and regulatory mechanism of TOL overproduction, an integrated analysis of metabolomics, genomics, and transcriptomics was performed. The results revealed that 1) both the Ehrlich pathway and tryptamine-dependent pathway were involved in S. cerevisiae TOL biosynthesis; 2) Trp increased TOL production by enhancing the Ehrlich pathway, in which the steps of transamination (including aminotransferase genes aro9, aat1, bat2 and his5) and decarboxylation (including decarboxylase genes aro10 and pdc5) played important roles. Of course, this process was assisted by amino acid permease genes agp1 and tat2, dihydrolipoyl dehydrogenase gene lpd1, and transcriptional activator gene aro80, etc.; 3) Phe restricted TOL biosynthesis by repressing the transcript levels of genes such as aat1, his5, aro10, pdc5 and aro80, thus interfering with the transamination and decarboxylation reactions; and 4) under sufficient Trp conditions, the de novo Trp biosynthetic pathway and central carbon metabolism (glycolysis, pentose phosphate pathway, and citrate cycle) of S. cerevisiae were weakened, while the content of some amino acids increased, which may be related to the promotion of yeast cell growth by Trp.ConclusionsIn this study, TOL production of S. cerevisiae was significantly improved, and our integrated multi-omics analyses have provided insights into the understanding of TOL over-accumulation, which will be useful for future production of TOL using metabolic engineering strategies.