To construct a superior microbial cell factory for chemical synthesis, a major challenge is to fully exploit cellular potential by identifying and engineering beneficial gene targets in sophisticated metabolic networks. Here, we take advantage of CRISPR interference (CRISPRi) and omics analyses to systematically identify beneficial genes that can be engineered to promote free fatty acids (FFAs) production in Escherichia coli. CRISPRi-mediated genetic perturbation enables the identification of 30 beneficial genes from 108 targets related to FFA metabolism. Then, omics analyses of the FFAs-overproducing strains and a control strain enable the identification of another 26 beneficial genes that are seemingly irrelevant to FFA metabolism. Combinatorial perturbation of four beneficial genes involving cellular stress responses results in a recombinant strain ihfAL−-aidB+-ryfAM−-gadAH−, producing 30.0 g L−1 FFAs in fed-batch fermentation, the maximum titer in E. coli reported to date. Our findings are of help in rewiring cellular metabolism and interwoven intracellular processes to facilitate high-titer production of biochemicals.

Electroactive microorganisms (EAMs) use bidirectional extracellular electron transfer (EET) pathways to exchange electrons with environments, enabling a diverse array of bio-electrochemical systems (BES). [1] These BES include microbial fuel cells (MFC) for electricity production from biomass and organic wastes, [2] electrochemical microbial biosensors for biotoxicity detection, [3] microbial desalination cells for seawater desalination, [4] microbial electrolysis cells for H 2 production, [5] and microbial electrosynthesis (MES) and photoelectrochemical biohybrid systems for the production of chemicals and fuels from CO 2 . [6] The EET efficiency is a dominating factor for the practical applications of BES. [7] Shewanella oneidensis MR-1, as a model EAM with contact-based and electron shuttle-based EET pathways, has been widely used in the EET study. [8] EET is however associated with multiple energy and materials metabolisms and cellular processes, for example, intracellular redox conditions, redox mediators, anaerobic carbon metabolism, and biofilm formation, etc. [9] This complexity makes it urgent to develop powerful genetic manipulation tools to engineer S. oneidensis MR-1 for improving the EET efficiency.Many gene expression and regulation toolbox have been developed in S. oneidensis MR-1, including plasmid expression toolkit [10] and CRISPR (clustered regularly interspaced short palindromic repeat)-mediated genome editing and regulation approaches. [11] A CRISPR-mediated base editing system (pCBEso) was recently developed in S. oneidensis MR-1, [12] in which C to T conversion in a 6-nt editing window could be achieved by the fusion protein of nCas9 (D10A) and cytidine deaminase rAPOBEC1. Base editing could achieve gene deactivation via mutating the CAG, CAA, CGA, TGG codons into premature stop codons (TAA, TAG, and TGA). Compared with the CRISPR-based recombination system that requires multiple components (two plasmids and ssDNA repair template), [11a] the base editing technologies are easier to operate, which avoid introduction of DNA double-strand breaks into the genome. However, to deactivate genes by base editing effectively, the premature stop codon should be introduced as close as possible Shewanella oneidensis MR-1, as a model electroactive microorganism (EAM) for extracellular electron transfer (EET) study, plays a key role in advancing practical applications of bio-electrochemical systems (BES). Efficient genome-level manipulation tools are vital to promote EET efficiency; thus, a powerful and rapid base editing toolbox in S. oneidensis MR-1 is developed. Firstly a CRISPR/ dCas9-AID base editor that shows a relatively narrow editing window restricted to the "−20 to −16" range upstream of the protospacer adjacent motif (PAM) is constructed. Cas9 is also confined by its native PAM requirement, NGG. Then to expand the editable scope, the sgRNA and the Cas-protein to broaden the editing window to "−22 to −9" upstream of the PAM are engineered, and the PAM field to NNN is opened up. Consequently, th...

1H NMR spectroscopy is often used to discriminate enantiomers of chiral analytes and determine their enantiomeric excess (ee) by various chiral auxiliaries. In reported research, these studies were mainly focused on chiral discriminantion of chiral analytes with only one chiral center. However, many chiral compounds possessing two or more chiral centers are often found in natural products, chiral drugs, products of asymmetric synthesis and biological systems. Therefore, it is necessary to investigate their chiral discrimination by effective chiral auxiliaries using 1H NMR spectroscopy. In this paper, a new class of tetraaza macrocyclic chiral solvating agents (TAMCSAs) with two amide (CONH), two amino (NH) and two phenolic hydroxyl (PhOH) groups has been designed and synthsized for chiral discrimination towards dipeptide derivatives with two chiral centers. These dipeptide derivatives are important chiral species because some of them are used as clinical drugs and special dietary supplements for treatment of human diseases, such as L-alanyl-L-glutamine and aspartame. The results show that these TAMCSAs have excellent chiral discriminating properties and offer multiple detection possibilities pertaining to 1H NMR signals of diagnostic split protons. The nonequivalent chemical shifts (up to 0.486 ppm) of various types of protons of these dipeptide derivatives were evaluated with the assistance of well-resolved 1H NMR signals in most cases. In addition, enantiomeric excesses (ee) of the dipeptide derivatives with different optical compositions have been calculated based on integration of well-separeted proton signals. At the same time, the possible chiral discriminating behaviors have been discussed by means of Job plots, ESI mass spectra and a proposed theoretical model of (±)-G1 with TAMCSA 1c. Additionally, the association constants of enantiomers of (±)-G5 with TAMCSA 1a were calculated by employing the nonlinear curve-fitting method.