BACKGROUND: Global transcription machinery engineering (gTME) is an effective approach employed in strain engineering to rewire gene expression and reshape cellular metabolic fluxes at the transcriptional level.
RESULTS: In this study, we utilized gTME to engineer the positive transcription factor, DegU, in the regulation network of major alkaline protease, AprE, in Bacillus pumilus. To validate its functionality when incorporated into the chromosome, we performed several experiments. First, three negative transcription factors, SinR, Hpr, and AbrB, were deleted to promote AprE synthesis. Second, several hyper-active DegU mutants, designated as DegU(hy), were selected using the fluorescence colorimetric method with the host of the Bacillus subtilis ΔdegSU mutant. Third, we integrated a screened degU(L113F) sequence into the chromosome of the Δhpr mutant of B. pumilus SCU11 to replace the original degU gene using a CRISPR/Cas9 system. Finally, based on transcriptomic and molecular dynamic analysis, we interpreted the possible mechanism of high-yielding and found that the strain produced alkaline proteases 2.7 times higher than that of the control strain (B. pumilus SCU11) in LB medium.
CONCLUSIONS: Our findings serve as a proof-of-concept that tuning the global regulator is feasible and crucial for improving the production performance of B. pumilus. Additionally, our study established a paradigm for gene function research in strains that are difficult to handle.

Due to the ability to regulate target metabolic pathways globally and dynamically, metabolic regulation systems composed of transcription factors have been widely used in metabolic engineering and synthetic biology. This review introduced the categories, action principles, prediction strategies, and related databases of transcription factors. Then, the application of global transcription machinery engineering technology and the transcription factor-based biosensors and quorum sensing systems are overviewed. In addition, strategies for optimizing the transcriptional regulatory tools\' performance by refactoring transcription factors are summarized. Finally, the current limitations and prospects of constructing various regulatory tools based on transcription factors are discussed. This review will provide theoretical guidance for the rational design and construction of transcription factor-based metabolic regulation systems.

Low isobutanol tolerance of Saccharomyces cerevisiae limits its application in isobutanol fermentation. Here, we used global transcription machinery engineering to screen mutants with higher isobutanol tolerance and elevated isobutanol titres. TATA-binding protein Spt15 was used as the target of global transcription machinery engineering for improvement of such complex phenotypes. A random mutagenesis library of S. cerevisiae TATA-binding protein Spt15 was constructed and subjected to screening under isobutanol stress. A mutant strain (denoted as spt15-3) with improved isobutanol tolerance was identified. There were three mutations of Spt15 in strain spt15-3, including deletion of A at position -132 nt upstream of initiation codon, insertion of G at position -65 nt upstream of initiation codon and a synonymous mutation at position 315 nt (T → C) downstream of initiation codon. We then metabolically engineered isobutanol synthesis in strains harbouring plasmids YCplac22 containing these Spt15 mutations. Delta integration was used to overexpress ILV3 gene, and 2μ plasmids carrying PGK1p-ILV2 and PGK1p-ARO10 were used to overexpress ILV2 and ARO10 genes. After 24-h micro-aerobic fermentation, Engi-3 produced 0·556 g l-1 isobutanol, which was 404% and 25·3% greater than isobutanol produced by control Engi-1 and engineered Engi-2, respectively. After 28 h, Engi-4 produced 0·459 g l-1 isobutanol, which was 315% and 3·2% greater than isobutanol produced Engi-1 and Engi-2, respectively. RNA-Seq-based transcriptome analysis shows that mutations of Spt15 in strain spt15-3 increased the expression of SPT15. Meanwhile, compared with strain Engi-3, the spt15-3 mutation downregulated the expression of genes involved in the TCA cycle and glyoxylic acid cycle, but increased the expression of genes related to cell stability. This work demonstrates that isobutanol tolerance and production of S. cerevisiae can be improved by engineering its TATA-binding protein Spt15. This study clarified the molecular mechanisms regulating isobutanol production and tolerance in S. cerevisiae.

BACKGROUND: Indigo is a color molecule with a long history of being used as a textile dye. The conventional production methods are facing increasing economy, sustainability and environmental challenges. Therefore, developing a green synthesis method converting renewable feedstocks to indigo using engineered microbes is of great research and application interest. However, the efficiency of the indigo microbial biosynthesis is still low and needs to be improved by proper metabolic engineering strategies.
RESULTS: In the present study, we adopted several metabolic engineering strategies to establish an efficient microbial biosynthesis system for converting renewable carbon substrates to indigo. First, a microbial co-culture was developed using two individually engineered E. coli strains to accommodate the indigo biosynthesis pathway, and the balancing of the overall pathway was achieved by manipulating the ratio of co-culture strains harboring different pathway modules. Through carbon source optimization and application of biosensor-assisted cell selection circuit, the indigo production was improved significantly. In addition, the global transcription machinery engineering (gTME) approach was utilized to establish a high-performance co-culture variant to further enhance the indigo production. Through the step-wise modification of the established system, the indigo bioproduction reached 104.3 mg/L, which was 11.4-fold higher than the parental indigo producing strain.
CONCLUSIONS: This work combines modular co-culture engineering, biosensing, and gTME for addressing the challenges of the indigo biosynthesis, which has not been explored before. The findings of this study confirm the effectiveness of the developed approach and offer a new perspective for efficient indigo bioproduction. More broadly, this innovative approach has the potential for wider application in future studies of other valuable biochemicals\' biosynthesis.

The regulation of single gene transcription level in the metabolic pathway is often failed to significantly improve the titer of the target product, and even leads to the imbalance of carbon/nitrogen metabolic network and cofactor network. Global transcription machinery engineering (gTME) can activate or inhibit the synergistic expression of multiple genes in specific metabolic pathways, so transcription factors with specific functions can be expressed according to different metabolic regulation requirements, thus effectively increasing the synthesis of target metabolites. In addition, maintaining intracellular redox balance through cofactor engineering can realize the self-balance of cofactors and promote the efficient synthesis of target products. In this study, we rebalanced the central carbon/nitrogen metabolism and redox metabolism of Corynebacterium glutamicum S9114 by gTME and redox cofactors engineering to promote the production of the nutraceutical N-acetylglucosamine (GlcNAc). Firstly, it was found that the overexpression of the transcription factor RamA can promote GlcNAc synthesis, and the titer was further improved to 16 g/L in shake flask by using a mutant RamA (RamAM). Secondly, a CRISPR interference (CRISPRi) system based on dCpf1 was developed and used to inhibit the expression of global negative transcriptional regulators of GlcNAc synthesis, which promoted the GlcNAc titer to 27.5 g/L. Thirdly, the cofactor specificity of the key enzymes in GlcNAc synthesis pathway was changed by rational protein engineering, and the titer of GlcNAc in shake flask was increased to 36.9 g/L. Finally, the production of GlcNAc was scaled up in a 50-L fermentor, and the titer reached 117.1 ± 1.9 g/L, which was 6.62 times that of the control group (17.7 ± 0.4 g/L), and the yield was increased from 0.19 g/g to 0.31 g/g glucose. The results obtained here highlight the importance of engineering the global regulation of central carbon/nitrogen metabolism and redox metabolism to improve the production performance of microbial cell factories.

During the last few years, the global transcription machinery engineering (gTME) technique has gained more attention as an effective approach for the construction of novel mutants. Genetic strategies (molecular biology methods) were utilized to get mutational for both genes (SPT15 and TAF23) basically existed in the Saccharomyces cerevisiae genome via screening the gTME approach in order to obtain a new mutant S. cerevisiae diploid strain. The vector pYX212 was utilized to transform these genes into the control diploid strain S. cerevisiae through the process of mating between haploids control strains S. cerevisiae (MAT-a [CICC 1374]) and (MAT-α [CICC 31144]), by using the oligonucleotide primers SPT15-EcoRI-FW/SPT15-SalI-RV and TAF23-SalI-FW/TAF23-NheI-RV, respectively. The resultant mutants were examined for a series of stability tests. This study showed how strong the effect of using strategic gTME with the importance of the modification we conducted in Error Prone PCR protocol by increasing MnCl2 concentration instead of MgCl2. More than ninety mutants we obtained in this study were distinguished by a high level production of bio-ethanol as compared to the diploid control strain.

A carbonyl reductase (cr) gene from Candida glabrata CBS138 has been heterologously expressed in cofactor regenerating E. coli host to convert Ethyl-4-chloro-3-oxobutanoate (COBE) into Ethyl-4-chloro-3-hydroxybutanoate (CHBE). The CR enzyme exhibited marked velocity at substrate concentration as high as 363mM with highest turnover number (112.77±3.95s-1). Solitary recombineering of such catalytic cell reproduced CHBE 161.04g/L per g of dry cell weight (DCW). Introduction of combinatorially engineered crp (crp*, F136I) into this heterologous E. coli host yielded CHBE 477.54g/L/gDCW. Furthermore, using nerolidol as exogenous cell transporter, the CHBE productivity has been towered to 710.88g/L/gDCW. The CHBE production has thus been upscaled to 8-12 times than those reported so far. qRT-PCR studies revealed that both membrane efflux channels such as acrAB as well as ROS scavenger genes such as ahpCF have been activated by engineering crp. Moreover, membrane protecting genes such as manXYZ together with solvent extrusion associated genes such as glpC have been upregulated inside mutant host. Although numerous proteins have been investigated to convert COBE to CHBE; this is the first approach to use engineering triad involving crp engineering, recombinant DNA engineering and transporter engineering together for improving cell performance during two-phase biocatalysis.

BACKGROUND: Escherichia coli has been explored as a platform host strain for biofuels production such as butanol. However, the severe toxicity of butanol is considered to be one major limitation for butanol production from E. coli. The goal of this study is therefore to construct butanol-tolerant E. coli strains and clarify the tolerance mechanisms.
RESULTS: A recombinant E. coli strain harboring σ(70) mutation capable of tolerating 2 % (v/v) butanol was isolated by the global transcription machinery engineering (gTME) approach. DNA microarrays were employed to assess the transcriptome profile of butanol-tolerant strain B8. Compared with the wild-type strain, 329 differentially expressed genes (197 up-regulated and 132 down-regulated) (p < 0.05; FC ≥ 2) were identified. These genes are involved in carbohydrate metabolism, energy metabolism, two-component signal transduction system, oxidative stress response, lipid and cell envelope biogenesis and efflux pump.
CONCLUSIONS: Several membrane-related proteins were proved to be involved in butanol tolerance of E. coli. Two down-regulated genes, yibT and yghW, were identified to be capable of affecting butanol tolerance by regulating membrane fatty acid composition. Another down-regulated gene ybjC encodes a predicted inner membrane protein. In addition, a number of up-regulated genes, such as gcl and glcF, contribute to supplement metabolic intermediates for glyoxylate and TCA cycles to enhance energy supply. Our results could serve as a practical strategy for the construction of platform E. coli strains as biofuel producer.