Enhanced Production of Pterostilbene in Escherichia coli Through Directed Evolution and Host Strain Engineering

Pterostilbene is a derivative of resveratrol with a higher bioavailability and biological activity, which shows antioxidant, anti-inflammatory, antitumor, and antiaging activities. Here, directed evolution and host strain engineering were used to improve the production of pterostilbene in Escherichia coli. First, the heterologous biosynthetic pathway enzymes of pterostilbene, including tyrosine ammonia lyase, p-coumarate: CoA ligase, stilbene synthase, and resveratrol O-methyltransferase, were successively directly evolved through error-prone polymerase chain reaction (PCR). Four mutant enzymes with higher activities of in vivo and in vitro were obtained. The directed evolution of the pathway enzymes increased the pterostilbene production by 13.7-fold. Then, a biosensor-guided genome shuffling strategy was used to improve the availability of the precursor L-tyrosine of the host strain E. coli TYR-30 used for the production of pterostilbene. A shuffled E. coli strain with higher L-tyrosine production was obtained. The shuffled strain harboring the evolved pathway produced 80.04 ± 5.58 mg/l pterostilbene, which is about 2.3-fold the highest titer reported in literatures.

Combination of Atmospheric and Room Temperature Plasma (ARTP) mutagenesis, Genome shuffling and Dimethyl sulfoxide (DMSO) feeding to improve FK506 production in Streptomyces tsukubaensis

FK506 is a clinically important macrocyclic polyketide with immunosuppressive activity produced by Streptomyces tsukubaensis . However, the production capacity of the strain is very low. To improve production, atmospheric and room temperature plasma (ARTP)mutagenesis was adopted to get the initial strains used in genome shuffling (GS). After three rounds of GS, S. tsukubaensis R3-C4 was the most productive strain, resultingin a FK506 concentration of 335 μg/mL, 2.6 times than that of the originalwild-type strain. Moreover, exogenous DMSO 4 % (v/v) addition could induce efflux of FK506 and increased FK506 production by 27.9% to 429 μg/mL . Finally, analyses of the differences in morphology, fermentation characteristics and specific gene expression levels between S. tsukubaensis R3-C4 and the wild-type strain revealed that R3-C4 strain: has hampered spore differentiation , thicker mycelia and more red pigment, which are likely related to the downregulation of bldD and cdgB expression. In addition, the expression levels of fkbO , fkbP , dahp , pccB and prpE all showed up-regulation at diverse degrees compared to the wild-type S. tsukubaensis . Overall, these results show that a combined approach involving classical random mutation and exogenous feeding canbe applied to increase FK506 biosynthesis andmay be applied also tothe improvement of other important secondary metabolites.