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

Abstract

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.

De Novo Pterostilbene Production from Glucose Using Modular Coculture Engineering in Escherichia coli

“…In our previous study, the de novo production of pterostilbene from glucose was based on bacterial monoculture. 13 Four necessary heterologous pathway enzymes, including tyrosine ammonia lyase (TAL) from Rhodotorula glutinis, p-coumarate:CoA ligase (4CL) from Arabidopsis thaliana, and stilbene synthase (STS), resveratrol O-methyltransferase (ROMT) from Vitis vinifera were optimized and introduced to an L-tyrosine overproducing strain E. coli TYR-14B1. The engineered strain contained the complete heterologous biosynthetic pathway necessary for pterostilbene biosynthesis, and the titer was 80.04 mg/L.…”

“…To implement this approach, a robust screening method is essential for identifying mutants with the desired phenotype. We have developed several metabolite-specific biosensors for in vivo monitoring shikimic acid, 33 L-tyrosine, 13 and 4hydroxyphenylacetic acid, 41 to facilitate the screening of the strain libraries derived from either ARTP mutagenesis or ep-WGS.…”

“…Error-Prone PCR-Based Whole Genome Shuffling. Errorprone PCR-based whole genome shuffling (ep-WGS) was performed according to previously described procedures 13,33,34 with some modifications. First, genomic DNA of E. coli MalCoA-30 mutants derived from ARTP mutagenesis was extracted and used as the template for error-prone whole genome amplification using 15-mer random primers named P R .…”

“…All the produced metabolites were eluted using a previously described gradient elution procedure. 10,13 Specifically, formic acid (0.1%) in water (solution A) and pure acetonitrile (solution B) were elution phases, maintaining a flow rate of 0. For relative quantification of the intracellular malonyl-CoA level in shake-flask fermentation, culture broth was centrifuged (12 000g, 5 min) to obtain the supernatant, and then OD 340 of the supernatant was measured.…”

Combining genetic engineering and bioprocess concepts for improved phenylpropanoid production

“…The stilbenoid resveratrol is commonly found in red wine, and is a popular supplement, while its' derivative pterostilbene exhibits increased bioavailability (Z. B. Yan et al, 2021).…”