Application of multiple strategies to enhance oleanolic acid biosynthesis by engineered Saccharomyces cerevisiae.

Oleanolic acid and its derivatives are widely used in the pharmaceutical, agricultural, cosmetic and food industries. Previous studies have shown that oleanolic acid production levels in engineered cell factories are low, which is why oleanolic acid is still widely extracted from traditional medicinal plants. To construct a highly efficient oleanolic acid production strain, rate-limiting steps were regulated by inducible promoters and the expression of key genes in the oleanolic acid synthetic pathway was enhanced. Subsequently, precursor pool expansion, pathway refactoring and diploid construction were considered to harmonize cell growth and oleanolic acid production. The multi-strategy combination promoted oleanolic acid production of up to 4.07 g/L in a 100 L bioreactor, which was the highest level reported.

A new l-serine binding orphan SerBP affects indole synthesis in Pantoea ananatis.

Indole is traditionally known as a metabolite of l-tryptophan and now as an important signaling molecule in bacteria, however, the understanding of its upstream synthesis regulation is very limited. Pantoea ananatis YJ76, a predominant diazotrophic endophyte isolated from rice (Oryza sativa), can produce indole to regulate various physiological and biochemical behaviors. We constructed a mutant library of YJ76 using the mTn5 transposon insertion mutation method, from which an indole-deficient mutant was screened out. Via high-efficiency thermal asymmetric interlaced PCR (hiTAIL-PCR), the transposon was determined to be inserted in a gene (RefSeq: WP014605468.1) of unknown function that is highly conserved at the intraspecific level. Bioinformatics analysis implied that the protein (Protein ID: WP089517194.1) encoded by the mutant gene is most likely to be a new orphan substrate-binding protein (SBP) for amino acid ABC transporters. Amino acid supplement cultivation experiments and surface plasmon resonance revealed that the protein could bind to l-serine (KD = 6.149 × 10-5 M). Therefore, the SBP was named as SerBP. This is the first case that a SBP responds to l-serine ABC transports. As a precursor of indole synthesis, the transmembrane transported l-serine was directly correlated with indole signal production and the mutation of serBP gene weakened the resistance of YJ76 to antibiotics, alkali, heavy metals, and starvation. This study provided a new paradigm for exploring the upstream regulatory pathway for indole synthesis of bacteria.