Dual genetic level modification engineering accelerate genome evolution of Corynebacterium glutamicum.

High spontaneous mutation rate is crucial for obtaining ideal phenotype and exploring the relationship between genes and phenotype. How to break the genetic stability of organisms and increase the mutation frequency has become a research hotspot. Here, we present a practical and controllable evolutionary tool (oMut-Cgts) based on dual genetic level modification engineering for Corynebacterium glutamicum. Firstly, the modification engineering of transcription and replication levels based on RNA polymerase α subunit and DNA helicase Cgl0854 as the 'dock' of cytidine deaminase (pmCDA1) significantly increased the mutation rate, proving that the localization of pmCDA1 around transient ssDNA is necessary for genome mutation. Then, the combined modification and optimization of engineering at dual genetic level achieved 1.02 × 104-fold increased mutation rate. The genome sequencing revealed that the oMut-Cgts perform uniform and efficient C:G→T:A transitions on a genome-wide scale. Furthermore, oMut-Cgts-mediated rapid evolution of C. glutamicum with stress (acid, oxidative and ethanol) tolerance proved that the tool has powerful functions in multi-dimensional biological engineering (rapid phenotype evolution, gene function mining and protein evolution). The strategies for rapid genome evolution provided in this study are expected to be applicable to a variety of applications in all prokaryotic cells.

Genomic and biological insights of bacteriophages JNUWH1 and JNUWD in the arms race against bacterial resistance.

The coevolution of bacteria and bacteriophages has created a great diversity of mechanisms by which bacteria fight phage infection, and an equivalent diversity of mechanisms by which phages subvert bacterial immunity. Effective and continuous evolution by phages is necessary to deal with coevolving bacteria. In this study, to better understand the connection between phage genes and host range, we examine the isolation and genomic characterization of two bacteriophages, JNUWH1 and JNUWD, capable of infecting Escherichia coli. Sourced from factory fermentation pollutants, these phages were classified within the Siphoviridae family through TEM and comparative genomic analysis. Notably, the phages exhibited a viral burst size of 500 and 1,000 PFU/cell, with latent periods of 15 and 20 min, respectively. They displayed stability over a pH range of 5 to 10, with optimal activity at 37°C. The complete genomes of JNUWH1 and JNUWD were 44,785 bp and 43,818 bp, respectively. Phylogenetic analysis revealed their close genetic relationship to each other. Antibacterial assays demonstrated the phages' ability to inhibit E. coli growth for up to 24 h. Finally, through laboratory-driven adaptive evolution, we successfully identified strains for both JNUWH1 and JNUWD with mutations in receptors specifically targeting lipopolysaccharides (LPS) and the lptD gene. Overall, these phages hold promise as additives in fermentation products to counter E. coli, offering potential solutions in the context of evolving bacterial resistance.

Application of modern synthetic biology technology in aromatic amino acids and derived compounds biosynthesis.

Aromatic amino acids (AAA) and derived compounds have enormous commercial value with extensive applications in the food, chemical and pharmaceutical fields. Microbial production of AAA and derived compounds is a promising prospect for its environmental friendliness and sustainability. However, low yield and production efficiency remain major challenges for realizing industrial production. With the advancement of synthetic biology, microbial production of AAA and derived compounds has been significantly facilitated. In this review, a comprehensive overview on the current progresses, challenges and corresponding solutions for AAA and derived compounds biosynthesis is provided. The most cutting-edge developments of synthetic biology technology in AAA and derived compounds biosynthesis, including CRISPR-based system, genetically encoded biosensors and synthetic genetic circuits, were highlighted. Finally, future prospects of modern strategies conducive to the biosynthesis of AAA and derived compounds are discussed. This review offers guidance on constructing microbial cell factory for aromatic compound using synthetic biology technology.

Utilizing 5' UTR Engineering Enables Fine-Tuning of Multiple Genes within Operons to Balance Metabolic Flux in Bacillus subtilis.

The application of synthetic biology tools to modulate gene expression to increase yield has been thoroughly demonstrated as an effective and convenient approach in industrial production. In this study, we employed a high-throughput screening strategy to identify a 5' UTR sequence from the genome of B. subtilis 168. This sequence resulted in a 5.8-fold increase in the expression level of EGFP. By utilizing the 5' UTR sequence to overexpress individual genes within the rib operon, it was determined that the genes ribD and ribAB serve as rate-limiting enzymes in the riboflavin synthesis pathway. Constructing a 5' UTR library to regulate EGFP expression resulted in a variation range in gene expression levels exceeding 100-fold. Employing the same 5' UTR library to regulate the expression of EGFP and mCherry within the operon led to a change in the expression ratio of these two genes by over 10,000-fold. So, employing a 5' UTR library to modulate the expression of the rib operon gene and construct a synthetic rib operon resulted in a 2.09-fold increase in riboflavin production. These results indicate that the 5' UTR sequence identified and characterized in this study can serve as a versatile synthetic biology toolkit for achieving complex metabolic network reconstruction. This toolkit can facilitate the fine-tuning of gene expression to produce target products.

Extraction of heavy metals from copper tailings by ryegrass (Lolium perenne L.) with the assistance of degradable chelating agents.

Heavy metal contamination is an urgent ecological governance problem in mining areas. In order to seek for a green and environmentally friendly reagent with better plant restoration effect to solve the problem of low efficiency in plant restoration in heavy metal pollution soil. In this study, we evaluated the effects of three biodegradable chelating agents, namely citric acid (CA), fulvic acid (FA) and polyaspartic acid (PASP), on the physicochemical properties of copper tailings, growth of ryegrass (Lolium perenne L.) and heavy metal accumulation therein. The results showed that the chelating agent application improved the physicochemical properties of copper tailings, increased the biomass of ryegrass and enriched more Cu and Cd in copper tailings. In the control group, the main existing forms of Cu and Cd were oxidizable state, followed by residual, weak acid soluble and reducible states. After the CA, FA or PASP application, Cu and Cd were converted from the residual and oxidizable states to the reducible and weak acid soluble states, whose bioavailability in copper tailings were thus enhanced. Besides, the chelating agent incorporation improved the Cu and Cd extraction efficiencies of ryegrass from copper tailings, as manifested by increased root and stem contents of Cu and Cd by 30.29-103.42%, 11.43-74.29%, 2.98-110.98% and 11.11-111.11%, respectively, in comparison with the control group. In the presence of multiple heavy metals, CA, FA or PASP showed selectivity regarding the ryegrass extraction of heavy metals from copper tailings. PCA analysis revealed that the CA-4 and PASP-7 treatment had great remediation potentials against Cu and Cd in copper tailings, respectively, as manifested by increases in Cu and Cd contents in ryegrass by 90.98% and 74.29% compared to the CK group.

Influence of conditioner and straw on the herbaceous plant-based phytoremediation copper tailings: a field trial at Liujiagou tailings pond, China.

A field trial was performed to carry out an enhanced phytoremediation technique for multi-metal contaminated copper tailings by Sudan grass (Sorghum Sudanese), ryegrass (Lolium perenne L.), and Bermuda grass (Cynodon dactylon), using conditioner (TH-LZ01) and straw combination into composite amendments as soil amendments, aimed to obtain the maximum of phytoremediation effect. The results showed that compared with untreated herbaceous plants, the application of conditioner and straw planted with herbaceous plants reduced the pH and conductivity and increased the organic matter and water content of the copper tailings to different degrees. With the addition of conditioner and straw, the DTPA-Cd, DTPA-Cu, DTPA-Pb, and DTPA-Zn contents in the copper tailings showed a decreasing trend compared with the untreated group. The herbaceous plants were promoted to reduce the percentage contents of acid soluble fractions Cd, Cu, Pb, and Zn and to increase the percentage contents of reducible, oxidizable, and residual fractions heavy metals (Cd, Cu, Pb, and Zn) in the copper tailings to different degrees. The contents of Cd, Cu, Pb, and Zn in the underground part of herbaceous plants were higher than those in the aboveground part, and the contents of Cd, Cu, Pb, and Zn in the aboveground part and underground part decreased after adding conditioner and straw, which indicated that the conditioner and straw inhibited the transport of heavy metals in the plant. Furthermore, the principal component analysis showed that the application of conditioner and straw with planting ryegrass had more potential for improving the physicochemical properties of copper tailings and reducing heavy metal toxicity, followed by Bermuda grass and Sudan grass.

Microbial production of branched chain amino acids: Advances and perspectives.

Branched-chain amino acids (BCAAs) such as L-valine, L-leucine, and L-isoleucine are widely used in food and feed. To comply with sustainable development goals, commercial production of BCAAs has been completely replaced with microbial fermentation. However, the efficient production of BCAAs by microorganisms remains a serious challenge due to their staggered metabolic networks and cell growth. To overcome these difficulties, systemic metabolic engineering has emerged as an effective and feasible strategy for the biosynthesis of BCAA. This review firstly summarizes the research advances in the microbial synthesis of BCAAs and representative engineering strategies. Second, systematic methods, such as high-throughput screening, adaptive laboratory evolution, and omics analysis, can be used to analyses the synthesis of BCAAs at the whole-cell level and further improve the titer of target chemicals. Finally, new tools and engineering strategies that may increase the production output and development direction of the microbial production of BCAAs are discussed.

Engineering serine hydroxymethyltransferases for efficient synthesis of L-serine in Escherichia coli.

L-serine is a high-value amino acid widely used in the food, medicine, and cosmetic industries. However, the low yield of L-serine has limited its industrial production. In this study, a cellular factory for efficient synthesis of L-serine was obtained by engineering the serine hydroxymethyltransferases (SHMT). Firstly, after screening the SHMT from Alcanivorax dieselolei by genome mining, a mutant AdSHMTE266M with high thermal stability was identified through rational design. Subsequently, an iterative saturating mutant library was constructed by using coevolutionary analysis, and a mutant AdSHMTE160L/E193Q with enzyme activity 1.35 times higher than AdSHMT was identified. Additionally, the target protein AdSHMTE160L/E193Q/E266M was efficiently overexpressed by improving its mRNA stability. Finally, combining the substrate addition strategy and system optimization, the optimized strain BL21/pET28a-AdSHMTE160L/E193Q/E266M-5'UTR-REP3S16 produced 106.06 g/L L-serine, which is the highest production to date. This study provides new ideas and insights for the engineering design of SHMT and the industrial production of L-serine.

Pre-optimization and one-step preparation of cascade enzymes system with broad substrates by model guidance: Application of chiral L-norvaline and L-phenylglycine biosynthesis.

Cascade biocatalyst systems with catalytic promiscuity can be used for synthesis of a class of chiral chemicals but the optimization of these systems by model guidance is poorly explored. In this study, a cascade system with broad substrate spectrum was characterized and simulated by kinetic model with substrates of DL-Norvaline (DL-Nor) and DL-Phenylglycine (DL-Phg) as examples. To evaluate the optimal cascade system, maximum accumulation of intermediate products and conversion rate in the process were investigated by simultaneous solution of the rate equations for varying enzyme quantities. According to the simulation results, the cascade system was optimized by regulating the expression of D-amino acid oxidase and formate dehydrogenase and was prepared by one-step. The conversion efficiency of DL-Nor and DL-Phg have been significantly improved compared with that of before optimization. Moreover, the total of L-Nor and L-Phg were reached 498.2 mM and 79.5 mM through a gradient fed-batch conversion strategy, respectively.

Combining protein and metabolic engineering strategies for high-level production of L-theanine in Corynebacterium glutamicum.

L-theanine is a natural non-protein amino acid with wide applications. Thus, a high yield of L-theanine production is required on an industrial scale. Herein, an efficient L-theanine-producing strain of Corynebacterium glutamicum was constructed by combining protein and metabolic engineering. Firstly, a γ-glutamylmethylamide synthetase from Paracoccus aminovorans (PaGMAS) was isolated and engineered by computer-aided design, the resulting mutant E179K/N105R improved L-theanine yield by 36.61 %. Subsequently, to increase carbon flux towards L-theanine production, the gene ggt which degrades L-theanine, the gene alaT which participated in L-alanine synthesis, and the gene NCgl1221 which encodes glutamate-exporting protein were deleted. Finally, ppk gene was overexpressed to enhance intracellular ATP production. The reprogramed strain produced 44.12 g/L L-theanine with a yield of 57.11 % and productivity of 1.16 g/L/h, which is the highest L-theanine titer reported by Corynebacterium glutamicum. This study provides an efficient and economical biosynthetic pathway for the industrial production of L-theanine.

High-efficient production of L-homoserine in Escherichia coli through engineering synthetic pathway combined with regulating cell division.

L-Homoserine is an important amino acid as a precursor in synthesizing many valuable products. However, the low productivity caused by slow L-homoserine production during active cell growth in fermentation hinders its potential applications. In this study, strategies of engineering the synthetic pathway combined with regulating cell division were employed in an L-homoserine-producing Escherichia coli strain for efficiently biomanufacturing L-homoserine. First, the flux-control genes in the L-homoserine degradation pathway were omitted to redistribute carbon flux. To drive more carbon flux into L-homoserine production, the phosphoenolpyruvate-pyruvate-oxaloacetate loop was redrawn. Subsequently, the cell division was engineered by using the self-regulated promoters to coordinate cell growth and L-homoserine production. The ultimate strain HOM23 produced 101.31 g/L L-homoserine with a productivity of 1.91 g/L/h, which presented the highest L-homoserine titer and productivity to date from plasmid-free strains. The strategies used in this study could be applied to constructing cell factories for producing other L-aspartate derivatives.

Construction of a plasmid-free L-leucine overproducing Escherichia coli strain through reprogramming of the metabolic flux.

BACKGROUND: L-Leucine is a high-value amino acid with promising applications in the medicine and feed industries. However, the complex metabolic network and intracellular redox imbalance in fermentative microbes limit their efficient biosynthesis of L-leucine. RESULTS: In this study, we applied rational metabolic engineering and a dynamic regulation strategy to construct a plasmid-free, non-auxotrophic Escherichia coli strain that overproduces L-leucine. First, the L-leucine biosynthesis pathway was strengthened through multi-step rational metabolic engineering. Then, a cooperative cofactor utilization strategy was designed to ensure redox balance for L-leucine production. Finally, to further improve the L-leucine yield, a toggle switch for dynamically controlling sucAB expression was applied to accurately regulate the tricarboxylic acid cycle and the carbon flux toward L-leucine biosynthesis. Strain LEU27 produced up to 55 g/L of L-leucine, with a yield of 0.23 g/g glucose. CONCLUSIONS: The combination of strategies can be applied to the development of microbial platforms that produce L-leucine and its derivatives.

Multidimensional engineering of Escherichia coli for efficient synthesis of L-tryptophan.

Development of microbial cell factory for L-tryptophan (L-trp) production has received widespread attention but still requires extensive efforts due to weak metabolic flux distribution and low yield. Here, the riboswitch-based high-throughput screening (HTS) platform was established to construct a powerful L-trp-producing chassis cell. To facilitate L-trp biosynthesis, gene expression was regulated by promoter and N-terminal coding sequences (NCS) engineering. Modules of degradation, transport and by-product synthesis related to L-trp production were also fine-tuned. Next, a novel transcription factor YihL was excavated to negatively regulate L-trp biosynthesis. Self-regulated promoter-mediated dynamic regulation of branch pathways was performed and cofactor supply was improved for further L-trp biosynthesis. Finally, without extra addition, the yield of strain Trp30 reached 42.5 g/L and 0.178 g/g glucose after 48 h of cultivation in 5-L bioreactor. Overall, strategies described here worked up a promising method combining HTS and multidimensional regulation for developing cell factories for products in interest.

Efficient 2-O-α-D-glucopyranosyl-sn-glycerol production by single whole-cell biotransformation through combined engineering and expression regulation with novel sucrose phosphorylase from Leuconostoc mesenteroides ATCC 8293.

2-O-α-D-glucopyranosyl-sn-glycerol (2-αGG) is a high value product with wide applications. Here, an efficient, safe and sustainable bioprocesses for 2-αGG production was designed. A novel sucrose phosphorylase (SPase) was firstly identified from Leuconostoc mesenteroides ATCC 8293. Subsequently, SPase mutations were processed with computer-aided engineering, of which the activity of SPaseK138C was 160% higher than that of the wild-type. Structural analysis revealed that K138C was a key functional residue moderating substrate binding pocket and thus influences catalytic activity. Furthermore, Corynebacterium glutamicum was employed to construct microbial cell factories along with ribosome binding site (RBS) fine-tuning and a two-stage substrate feeding control strategy. The maximum production of 2-αGG by these combined strategies reached 351.8 g·L-1 with 98% conversion rate from 1.4 M sucrose and 3.5 M glycerol in a 5-L bioreactor. This was one of the best performance reported in single-cell biosynthesis of 2-αGG, which paved effective ways for industrial-scale preparation of 2-αGG.

Direct evolution of riboflavin kinase significantly enhance flavin mononucleotide synthesis by design and optimization of flavin mononucleotide riboswitch.

Flavin mononucleotide (FMN) is the active form of riboflavin. It has a wide range of application scenarios in the pharmaceutical and food additives. However, there are limitations in selecting generic high-throughput screening platforms that improve the properties of enzymes. First, the biosensor in response to FMN concentration was constructed using the FMN riboswitch and confirmed the function of this sensor. Next, the FMN binding site of the sensor was saturated with a mutation that increased its fluorescence range by approximately 127%. Then, the biosensor and the base editing system based on T7RNAP were combined to construct a platform for rapid mutation and screening of riboflavin kinase gene ribC mutants. The mutants screened using this platform increased the yield of FMN by 8-fold. These results indicate that the high-throughput screening platform can rapidly and effectively improve the activity of target enzymes, and provide a new route for screening industrial enzymes.

Variants of a putative baseplate wedge protein extend the host range of Pseudomonas phage K8.

BACKGROUND: Narrow host range is a major limitation for phage applications, but phages can evolve expanded host range through adaptations in the receptor-binding proteins. RESULTS: Here, we report that Pseudomonas phage K8 can evolve broader host range and higher killing efficiency at the cost of virion stability. Phage K8 host range mutant K8-T239A carries a mutant version of the putative baseplate wedge protein GP075, termed GP075m. While phage K8 adsorbs to hosts via the O-specific antigen of bacterial LPS, phage K8-T239A uses GP075m to also bind the bacterial core oligosaccharide, enabling infection of bacterial strains resistant to K8 infection due to modified O-specific antigens. This mutation in GP075 also alters inter-protein interactions among phage proteins, and reduces the stability of phage particles to environmental stressors like heat, acidity, and alkalinity. We find that a variety of mutations in gp075 are widespread in K8 populations, and that the gp075-like genes are widely distributed among the domains of life. CONCLUSION: Our data show that a typical life history tradeoff occurs between the stability and the host range in the evolution of phage K8. Reservoirs of viral gene variants may be widely present in phage communities, allowing phages to rapidly adapt to any emerging environmental stressors. Video Abstract.

Efficient biosynthesis of (R)-mandelic acid from styrene oxide by an adaptive evolutionary Gluconobacter oxydans STA.

BACKGROUND: (R)-mandelic acid (R-MA) is a highly valuable hydroxyl acid in the pharmaceutical industry. However, biosynthesis of optically pure R-MA remains significant challenges, including the lack of suitable catalysts and high toxicity to host strains. Adaptive laboratory evolution (ALE) was a promising and powerful strategy to obtain specially evolved strains. RESULTS: Herein, we report a new cell factory of the Gluconobacter oxydans to biocatalytic styrene oxide into R-MA by utilizing the G. oxydans endogenous efficiently incomplete oxidization and the epoxide hydrolase (SpEH) heterologous expressed in G. oxydans. With a new screened strong endogenous promoter P12780, the production of R-MA was improved to 10.26 g/L compared to 7.36 g/L of using Plac. As R-MA showed great inhibition for the reaction and toxicity to cell growth, adaptive laboratory evolution (ALE) strategy was introduced to improve the cellular R-MA tolerance. The adapted strain that can tolerate 6 g/L R-MA was isolated (named G. oxydans STA), while the wild-type strain cannot grow under this stress. The conversion rate was increased from 0.366 g/L/h of wild type to 0.703 g/L/h by the recombinant STA, and the final R-MA titer reached 14.06 g/L. Whole-genome sequencing revealed multiple gene-mutations in STA, in combination with transcriptome analysis under R-MA stress condition, we identified five critical genes that were associated with R-MA tolerance, among which AcrA overexpression could further improve R-MA titer to 15.70 g/L, the highest titer reported from bulk styrene oxide substrate. CONCLUSIONS: The microbial engineering with systematic combination of static regulation, ALE, and transcriptome analysis strategy provides valuable solutions for high-efficient chemical biosynthesis, and our evolved G. oxydans would be better to serve as a chassis cell for hydroxyl acid production.

Effect of composite amendments on physicochemical properties of copper tailings repaired by herbaceous plants.

Phytoremediation is considered to be the most environmentally friendly green restoration technology for dealing with mine waste. Adding amendments can improve the substrate environment for plant growth and enhance remediation efficiency. Herbaceous plants have become the preferred species for vegetation restoration in abandoned mines because of their fast greening and simple management. After 8 weeks of pot experiments in the early stage, it was shown that the plant height and fresh weight of the plants treated with 5% conditioner and 0.5% straw (C2S2) were significantly higher than those of other treatments. Considering that, in this paper, to explore the effect of composite amendments on physicochemical properties of copper tailings repaired by herbaceous plants, the untreated copper tailings were employed as the control group, whereas copper tailings repaired by ryegrass (Lolium perenne L.), vetiver grass (Chrysopogon zizanioides L.), and tall fescue (Festuca arundinacea) with or without conditioners and straw combination into the compound amendments were taken separately as the test group. After 6 months of planting, the pH, electrical conductivity, water content, available potassium, organic matter, total nitrogen, and available phosphorus in the main physical and chemical properties of copper tailings in each experimental area were analyzed. The results showed that the electrical conductivity, organic matter, and total nitrogen content of copper tailings were improved to a certain extent by planting plants without treatment. Meanwhile, compared with the control group, all indexes of planting plants showed an upward trend after adding composite amendments. Among them, pH, water content, and available potassium content of copper tailings were enhanced more obviously. Furthermore, as discovered from the gray correlation analysis results, vetiver grass planted with composite amendments has the best comprehensive effect of improving the physicochemical properties of copper tailings, followed by tall fescue and ryegrass.

Improving prodigiosin production by transcription factor engineering and promoter engineering in Serratia marcescens.

Prodigiosin (PG), a red linear tripyrrole pigment produced by Serratia marcescens, has attracted attention due to its immunosuppressive, antimicrobial, and anticancer properties. Although many studies have been used to dissect the biosynthetic pathways and regulatory network of prodigiosin production in S. marcescens, few studies have been focused on improving prodigiosin production through metabolic engineering in this strain. In this study, transcription factor engineering and promoter engineering was used to promote the production of prodigiosin in S. marcescens JNB5-1. Firstly, through construing of a Tn5G transposon insertion library of strain JNB5-1, it was found that the DNA-binding response regulator BVG89_19895 (OmpR) can promote prodigiosin synthesis in this strain. Then, using RNA-Seq analysis, reporter green fluorescent protein analysis and RT-qPCR analysis, the promoter P17 (P RplJ ) was found to be a strong constitutive promoter in strain JNB5-1. Finally, the promoter P17 was used for overexpressing of prodigiosin synthesis activator OmpR and PsrA in strain JNB5-1 and a recombinant strain PG-6 was obtained. Shake flask analysis showed that the prodigiosin titer of this strain was increased to 10.25 g/L, which was 1.62-times that of the original strain JNB5-1 (6.33 g/L). Taken together, this is the first well-characterized constitutive promoter library from S. marcescens, and the transcription factor engineering and promoter engineering can be also useful strategies to improve the production of other high value-added products in S. marcescens.

High-level production of L-valine in Escherichia coli using multi-modular engineering.

L-valine is a valuable amino acid in mammals that is used as the main component of feed additives. The low efficiency of the fermentation titer limits the industrial application of L-valine. Here, an L-valine-producing strain of Escherichia coli was obtained using a multi-modular strategy. Initially, a chassis strain was generated by mutagenesis and high-throughput screening. The L-valine biosynthetic pathway and transport module were modified to improve the L-valine titer. Subsequently, the transcription factors associated with L-valine biosynthesis were investigated. Overexpression of PdhR and inhibition of the expression of RpoS promoted L-valine synthesis. Finally, the NADPH supply was enhanced after the introduction of the heterologous Entner-Doudoroff (ED) pathway from Zymomonas mobilis. The strain VAL38 produced 92 g/L L-valine in a 5-L bioreactor with a yield of 0.34 g/g glucose. This strategy is provided as a reference for improving the production performance of cell factories for L-valine and its derivatives.