Fluorescence-based high-throughput screening approaches facilitate the discovery of enzymes and microorganisms for polyethylene terephthalate (PET) depolymerization and recycling. However, the traditional method of activating the fluorescence signal by cleaving the ester bond on small molecule probes has limited ability in detecting enzymatic activity towards polymeric substrates. This study proposes a novel fluorescence-based screening strategy that detects the release of sulfhydryl groups during the depolymerization of pseudo-PET polythioesters by polyesterases. The strategy successfully identifies the polyester-depolymerizing activity of leaf-branch compost cutinase (LCC ICCG), while porcine liver esterase (PLE) only hydrolyses small molecular substrates. When combined with a droplet microfluidic system, the strategy enables high-throughput screening of LCC ICCG. The study also demonstrates that screening for polyester-depolymerizing bacteria can be performed via a microplate reader platform. The new screening approach offers an efficient method for identifying enzymes and microbial resources for depolymerizing polyester-like plastics.

Butyl acetate has shown wide attention in food, cosmetics, medicine and biofuel sectors. These short chain fatty acid esters can be produced by either chemical or biological synthetic process with corresponding alcohols and acids. Currently, biosynthesis of short chain fatty acid esters, such as butyl butyrate through microbial fermentation systems has been achieved; however, few studies regarding biosynthesis of butyl acetate were reported. In this study, three proof-of-principle strategies for the one-pot butyl acetate production from glucose by microbial fermentation was designed and evaluated. (1) 7.3 g/L of butyl acetate was synthesized by butanol producing Clostridium acetobutylicum NJ4 with the supplementation of exogenous acetic acid; (2) With the addition of butanol, 5.76 g/L of butyl acetate can be synthesized by acetate producing Actinobacillus succinogenes130z (ΔpflA) with the supplementation of exogenous butanol; (3) Microbial co-culture of C. acetobutylicum NJ4 and A. succinogenes130z (ΔpflA) can directly produce 2.2 g/L of butyl acetate from glucose, representing the first study in terms of butyl acetate production by using microbial co-culture system. Through the immobilization of A. succinogenes130z (ΔpflA), butyl acetate production was further improved to 2.86 g/L. These strategies may be extended to the biosynthesis of a wide range of esters, especially to some longer chain ones.

Consolidated bioprocessing (CBP) has been widely adopted as a cost-effective strategy for the bioconversion of lignocellulosic biomass into bio-chemicals. Microbial consortium can complete the complex CBP processes through the cooperation of different microorganisms. In this study, a synthetic microbial consortium was designed, which is composed of a hemicellulase-producing bacterium Thermoanaerobacterium thermosaccharolyticum and succinic acid production specialist Actinobacillus succinogenes 130Z. The simultaneous conversion of xylose hydrolyzed by T. thermosaccharolyticum could maintain a high hydrolyzing rate, which would facilitate succinic acid production by A. succinogenes 130Z. After process optimization, 32.50 g/L of succinic acid with yield of 0.41 g/g was obtained from 80 g/L xylan through CBP, representing the highest succinic acid production directly from hemicellulose materials. In addition, 12.51 g/L of succinic acid was directly produced from 80 g/L of corn cob. The above results demonstrated that this CBP based microbial co-cultivation system had great potential to convert lignocellulosic biomass into various bio-chemicals.

A unique Meyerozyma sp. strain YLG18 was obtained in this study, which was capable of producing 2-phenylethanol through both Ehrlich and Shikimate pathways. Response surface methodology (RSM) was implemented to improve the maximum 2-PE production. At optimized conditions: temperature, 24.7℃; initial glucose, 63.54 g/L; initial L-phe, 10.70 g/L, 2-PE production was increased to 2.55 g/L, leading to 104% increase compared to the pre-optimized one. In situ product recovery (ISPR) could further help improve the final 2-PE production to 3.20 g/L with fatty acid methyl ester as the extractant, representing the highest 2-PE production by using Meyerozyma sp.. Furthermore, genes involved in 2-PE synthesis were identified and their expression levels between Shikimate pathway and Ehrlich pathway were compared. Based on the genomic and transcriptional analysis, a penta-functional enzyme AroM in Shikimate pathway and an aspartate aminotransferase (AAT) with the potential to convert phenylalanine into phenylpyruvate in Ehrlich pathway were identified. These findings would help broaden our knowledge and add to the pool of known 2-PE generating microbes and genes.