Identification of crucial roles of transcription factor IhfA on high production of free fatty acids in .

Transcription factor engineering has unique advantages in improving the performance of microbial cell factories due to the global regulation of gene transcription. Omics analyses and reverse engineering enable learning and subsequent incorporation of novel design strategies for further engineering. Here, we identify the role of the global regulator IhfA for overproduction of free fatty acids (FFAs) using CRISPRi-facilitated reverse engineering and cellular physiological characterization.

Enhanced acetate utilization for value-added chemicals production in Yarrowia lipolytica by integration of metabolic engineering and microbial electrosynthesis.

The limited supply of reducing power restricts the efficient utilization of acetate in Yarrowia lipolytica. Here, microbial electrosynthesis (MES) system, enabling direct conversion of inward electrons to NAD(P)H, was used to improve the production of fatty alcohols from acetate based on pathway engineering. First, the conversion efficiency of acetate to acetyl-CoA was reinforced by heterogenous expression of ackA-pta genes.

Recent advances in enrichment, isolation, and bio-electrochemical activity evaluation of exoelectrogenic microorganisms.

Exoelectrogenic microorganisms (EEMs) catalyzed the conversion of chemical energy to electrical energy via extracellular electron transfer (EET) mechanisms, which underlay diverse bio-electrochemical systems (BES) applications in clean energy development, environment and health monitoring, wearable/implantable devices powering, and sustainable chemicals production, thereby attracting increasing attentions from academic and industrial communities in the recent decades. However, knowledge of EEMs is still in its infancy as only ∼100 EEMs of bacteria, archaea, and eukaryotes have been identified, motivating the screening and capture of new EEMs. This review presents a systematic summarization on EEM screening technologies in terms of enrichment, isolation, and bio-electrochemical activity evaluation.

Highly efficient multiplex base editing: One-shot deactivation of eight genes in MR-1.

Obtaining electroactive microbes capable of efficient extracellular electron transfer is a large undertaking for the scalability of bio-electrochemical systems. Inevitably, researchers need to pursue the co-modification of multiple genes rather than expecting that modification of a single gene would make a significant contribution to improving extracellular electron transfer rates. Base editing has enabled highly-efficient gene deactivation in model electroactive microbe MR-1.

Genome Editing by CRISPR/Cas12 Recognizing AT-Rich PAMs in MR-1.

Homologous recombination-mediated genomic editing is urgently needed to obtain high-performance chassis of electroactive microorganisms. However, the existing tools cannot meet the requirement of genome-wide editing in . Here, we develop different CRISPR-Cas systems that are ideal to be employed in AT-rich sequences as the supplements to Cas9.

Coupling riboflavin de novo biosynthesis and cytochrome expression for improving extracellular electron transfer efficiency in Shewanella oneidensis.

Shewanella oneidensis MR-1, as a model exoelectrogen with divergent extracellular electron transfer (EET) pathways, has been widely used in microbial fuel cells (MFCs). The electron transfer rate is largely determined by riboflavin (RF) and c-type cytochromes (c-Cyts). However, relatively low RF production and inappropriate amount of c-Cyts substantially impede the capacity of improving the EET rate.

Transcriptome Analysis to Identify Crucial Genes for Reinforcing Flavins-Mediated Extracellular Electron Transfer in .

Flavins serve as the electron mediators in , determining the extracellular electron transfer (EET) rate. Currently, metabolic engineering of flavins biosynthetic pathway has been studied for improving EET. However, the cellular response triggered by flavins that contribute to EET remains to be elucidated.

Type I-F CRISPR-PAIR platform for multi-mode regulation to boost extracellular electron transfer in .

Bio-electrochemical systems are based on extracellular electron transfer (EET), whose efficiency relates to the expression level of numerous genes. However, the lack of multi-functional tools for gene activation and repression hampers the enhancement of EET in electroactive microorganisms (EAMs). We thus develop a type I-F CRISPR/PaeCascade-RpoD-mediated activation and inhibition regulation (CRISPR-PAIR) platform in the model EAM, MR-1.

CRISPR/dCas9-RpoD-Mediated Simultaneous Transcriptional Activation and Repression in MR-1.

Extracellular electron transfer (EET) of electroactive microorganisms (EAMs) is the dominating factor for versatile applications of bio-electrochemical systems. MR-1 is one of the model EAMs for the study of EET, which is associated with a variety of cellular activities. However, due to the lack of a transcriptional activation tool, regulation of multiple genes is labor-intensive and time-consuming, which hampers the advancement of improving the EET efficiency in .

Non-homologous End Joining-Mediated Insertional Mutagenesis Reveals a Novel Target for Enhancing Fatty Alcohols Production in .

Non-homologous end joining (NHEJ)-mediated integration is effective in generating random mutagenesis to identify beneficial gene targets in the whole genome, which can significantly promote the performance of the strains. Here, a novel target leading to higher protein synthesis was identified by NHEJ-mediated integration that seriously improved fatty alcohols biosynthesis in . One batch of strains transformed with fatty acyl-CoA reductase gene () showed significant differences (up to 70.

Development of Whole Genome-Scale Base Editing Toolbox to Promote Efficiency of Extracellular Electron Transfer in Shewanella oneidensis MR-1.

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.

Establishment of genomic library technology mediated by non-homologous end joining mechanism in Yarrowia lipolytica.

Genomic variants libraries are conducive to obtain dominant strains with desirable phenotypic traits. The non-homologous end joining (NHEJ), which enables foreign DNA fragments to be randomly integrated into different chromosomal sites, shows prominent capability in genomic libraries construction. In this study, we established an efficient NHEJ-mediated genomic library technology in Yarrowia lipolytica through regulation of NHEJ repair process, employment of defective Ura marker and optimization of iterative transformations, which enhanced genes integration efficiency by 4.

[Construction of conjugated polymer-exoelectrogen hybrid bioelectrodes and applications in microbial fuel cells].

Microbial fuel cell (MFC) is a bioelectrochemical device, that enables simultaneous wastewater treatment and energy generation. However, a few issues such as low output power, high ohmic internal resistance, and long start-up time greatly limit MFCs' applications. MFC anode is the carrier of microbial attachment, and plays a key role in the generation and transmission of electrons.

Microbial extracellular electron transfer and strategies for engineering electroactive microorganisms.

Electroactive microorganisms (EAMs) are ubiquitous in nature and have attracted considerable attention as they can be used for energy recovery and environmental remediation via their extracellular electron transfer (EET) capabilities. Although the EET mechanisms of Shewanella and Geobacter have been rigorously investigated and are well characterized, much less is known about the EET mechanisms of other microorganisms. For EAMs, efficient EET is crucial for the sustainable economic development of bioelectrochemical systems (BESs).

High production of triterpenoids in through manipulation of lipid components.

Background: Lupeol exhibits novel physiological and pharmacological activities, such as anticancer and immunity-enhancing activities. However, cytotoxicity remains a challenge for triterpenoid overproduction in microbial cell factories. As lipophilic and relatively small molecular compounds, triterpenes are generally secreted into the extracellular space.

sRNA-Based Screening Chromosomal Gene Targets and Modular Designing for High-Titer Production of Aglycosylated Immunoglobulin G.

The production of the aglycosylated immunoglobulin G (IgG) in has received wide interest for its analytical and therapeutic applications. To enhance the production titer of IgG, we first used synthetic sRNAs to perform a systematical analysis of the gene expression in the translational level in the glycolytic pathway (module 1) and the tricarboxylic acid (TCA) cycle (module 2) to reveal the critical genes for the efficient IgG production. Second, to provide sufficient amino acid precursors for the protein biosynthesis, amino acid biosynthesis pathways (module 3) were enhanced to facilitate the IgG production.

Synthetic sRNA-Based Engineering of Escherichia coli for Enhanced Production of Full-Length Immunoglobulin G.

Production of monoclonal antibodies (mAbs) receives considerable attention in the pharmaceutical industry. There has been an increasing interest in the expression of mAbs in Escherichia coli for analytical and therapeutic applications in recent years. Here, a modular synthetic biology approach is developed to rationally engineer E.

Enhancing surfactin production by using systematic CRISPRi repression to screen amino acid biosynthesis genes in Bacillus subtilis.

Background: Surfactin is a cyclic lipopeptide that is of great industrial use owing to its extraordinary surfactant power and antimicrobial, antiviral, and antitumor activities. Surfactin is synthesized by a condensation reaction in microbes, which uses fatty acids and four kinds of amino acids (L-glutamate, L-aspartate, L-leucine and L-valine) as precursors. Surfactin biosynthesis could be improved by increasing the supply of fatty acids; however, the effect of the regulation of amino acid metabolism on surfactin production was not yet clear.

Boosting the biosynthesis of betulinic acid and related triterpenoids in Yarrowia lipolytica via multimodular metabolic engineering.

Background: Betulinic acid is a pentacyclic lupane-type triterpenoid and a potential antiviral and antitumor drug, but the amount of betulinic acid in plants is low and cannot meet the demand for this compound. Yarrowia lipolytica, as an oleaginous yeast, is a promising microbial cell factory for the production of highly hydrophobic compounds due to the ability of this organism to accumulate large amounts of lipids that can store hydrophobic products and supply sufficient precursors for terpene synthesis. However, engineering for the heterologous production of betulinic acid and related triterpenoids has not developed as systematically as that for the production of other terpenoids, thus the production of betulinic acid in microbes remains unsatisfactory.

Engineering Microbial Consortia for High-Performance Cellulosic Hydrolyzates-Fed Microbial Fuel Cells.

Microbial fuel cells (MFCs) are eco-friendly bio-electrochemical reactors that use exoelectrogens as biocatalyst for electricity harvest from organic biomass, which could also be used as biosensors for long-term environmental monitoring. Glucose and xylose, as the primary ingredients from cellulose hydrolyzates, is an appealing substrate for MFC. Nevertheless, neither xylose nor glucose can be utilized as carbon source by well-studied exoelectrogens such as .

A Synthetic Plasmid Toolkit for MR-1.

MR-1 is a platform microorganism for understanding extracellular electron transfer (EET) with a fully sequenced and annotated genome. In comparison to other model microorganisms such as , the available plasmid parts (such as promoters and replicons) are not sufficient to conveniently and quickly fine-tune the expression of multiple genes in MR-1. Here, we constructed and characterized a plasmid toolkit that contains a set of expression vectors with a combination of promoters, replicons, antibiotic resistance genes, and an RK2 origin of transfer () cassette, in which each element can be easily changed by fixed restriction enzyme sites.

Author Correction: Precise control of SCRaMbLE in synthetic haploid and diploid yeast.

The original version of this Article omitted a declaration from the Competing Interests statement, which should have included the following: 'J.D.B.

Modular Pathway Engineering of Bacillus subtilis To Promote De Novo Biosynthesis of Menaquinone-7.

Menaquinone-7 (MK-7), a valuable vitamin K, plays an important role in the prevention of osteoporosis and cardiovascular calcification. We chose B. subtilis 168 as the chassis for the modular metabolic engineering design to promote the biosynthesis of MK-7.