DNA-guided cell-free protein synthesis using a minimal set of purified components has emerged as a versatile platform in constructive biology. The E. coli-based PURE (Protein synthesis Using Recombinant Elements) system offers the basic protein synthesis factory in a prospective minimal cell relying on extant molecules. However, it becomes urgent to improve the system's performance, and to build a mechanistic computational model that can help interpret and predict gene expression dynamics. Herein, we utilized all three commercially available PURE system variants: PURExpress, PUREfrex and PUREfrex2.0. We monitored apparent kinetics of mRNA and protein synthesis by fluorescence spectroscopy at different concentrations of DNA template. Analysis of polysome distributions by atomic force microscopy, combined with a stochastic model of translation, revealed inefficient usage of ribosomes, consistent with the idea that translation initiation is a limiting step. This preliminary dataset was used to formulate hypotheses regarding possible mechanisms impeding robust gene expression. Next, we challenged these hypotheses by devising targeted experiments aimed to alleviate the current limitations of PUREfrex. We identified depletion of key initiation factors by translationally inactive mRNA as a possible inhibitory mechanism. This adverse process could partly be remedied by targeted mRNA degradation, whereas addition of more IFs and of the hrpA RNA helicase had no substantial effects. Moreover, depletion of tRNAs as peptidyl-tRNAs can become limiting in PUREfrex (but not in PURExpress), which can be alleviated by addition of peptidyl-tRNAhydrolase (PTH). We attempted to build a new model for PURE system dynamics integrating all experimental observations. Although a satisfying global fit can be obtained in specific conditions (with PTH), a unifying system's level model is still missing.

Pectin-rich biomasses, such as citrus peel and sugar beet pulp, hold promise as inexpensive feedstocks for microbial fermentations as enzymatic hydrolysis of their component polysaccharides can be accomplished inexpensively to yield high concentrations of fermentable sugars and d-galacturonic acid (d-galUA). In this study, we tackle a number of challenges associated with engineering a microbial strain to convert pectin-rich hydrolysates into commodity and specialty chemicals. First, we engineer d-galUA utilization into yeast, Saccharomyces cerevisiae. Second, we identify that the mechanism of d-galUA uptake into yeast is mediated by hexose transporters and that consumption of d-galUA is inhibited by d-glucose. Third, we enable co-utilization of d-galUA and d-glucose by identifying and expressing a heterologous transporter, GatA, from Aspergillus niger. Last, we demonstrate the use of this transporter for production of the platform chemical, meso-galactaric acid, directly from industrial Navel orange peel waste.

Summary We staged the transfer of the aurofusarin and bikaverin biosynthetic gene clusters (BGCs) to Aspergillus nidulans with the aim of gaining functional insights into dynamics immediately following a horizontal gene transfer (HGT) event. While the introduction of both BGCs resulted in the production of detectable pathway metabolites in A. nidulans, the transferred aurofusarin BGC formed dimeric shunt products instead of aurofusarin. This was linked to low transcription of the cluster activator and insufficient activity of tailoring enzymes, demonstrating how a shift of the pathway bottleneck after HGT can result in metabolic innovation. The transferred bikaverin BGC readily produced bikaverin, providing a model system for studying the conservation of regulatory responses to environmental cues. Conserved PacC‐mediated pH regulation of the bikaverin BGC was observed between original host Fusarium fujikuroi and A. nidulans. Contrary to strong nitrogen responses described in other hosts, the BGC appeared unresponsive to environmental nitrogen in A. nidulans. While F. fujikuroi and A. nidulans both form chlamydospore‐like structures when exposed to ralsolamycin, specific induction of the bikaverin BGC was not observed in A. nidulans. We propose that the presence of compatible cis‐regulatory elements in BGCs facilitates regulatory conservation after transfer, without which the chromosomal context would dictate expression.