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.

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.

Supramolecular crystals of Mn(15-crown-5)(MnCl4)(DMF) with dielectric phase transition, high quantum yield and phase transition-induced luminescence enhancement behavior.

The supramolecular crystals, Mn(15-crown-5)(MnCl4)(DMF), (1; 15-crown-5 = 1,4,7,10,13-pentaoxacyclopentadecane), were synthesized via a self-assembly strategy under ambient conditions. Comprehensive characterization of the crystals involved microanalysis for C, H, and N elements, thermogravimetric (TG) analysis, differential scanning calorimetry (DSC) and single-crystal X-ray diffraction techniques. The results reveal that 1 undergoes a two-step thermotropic and isostructural phase transition at around 217 K and 351 K upon heating. All three phases belong to the same space group (P212121) with analogous cell parameters. These two phase transitions primarily involve the thermally activated ring rotational dynamics of the 15-crown-5 molecule, with only the transition at ca. 351 K being associated with a dielectric anomaly. 1 exhibits intense luminescence with a peak at ∼600 nm and a high quantum yield of 68%. The mechanisms underlying this intense luminescence are likely linked to low-symmetry ligand fields. Additionally, 1 displays phase transition-induced luminescence enhancement behavior, and the possible mechanism is further 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.

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.

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.

Combinatorial protein engineering and transporter engineering for efficient synthesis of L-Carnosine in Escherichia coli.

L-Carnosine has various physiological functions and is widely used in cosmetics, medicine, food additives, and other fields. However, the yield of L-Carnosine obtained by biological methods is far from the level of industrial production. Herein, a cell factory for efficient synthesis of L-Carnosine was constructed based on transporter engineering and protein engineering. Firstly, a dipeptidase (SmpepD) was screened from Serratia marcescens through genome mining to construct a cell factory for synthesizing L-Carnosine. Subsequently, through rationally designed SmPepD, a double mutant T168S/G148D increased the L-Carnosine yield by 41.6% was obtained. Then, yeaS, a gene encoding the exporter of L-histidine, was deleted to further increase the production of L-Carnosine. Finally, L-Carnosine was produced by one-pot biotransformation in a 5 L bioreactor under optimized conditions with a yield of 133.2 mM. This study represented the highest yield of L-Carnosine synthesized in microorganisms and provided a biosynthetic pathway for the industrial production of L-Carnosine.

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.

Phase transition and ionic conduction enhancement induced by co-doping of LiI and MnI2 in a one-dimensional lead iodide perovskite.

Herein, we demonstrated the unique advantage of a mechanochemical reaction to prepare a salt with hard and soft acid and base ions concurrently by solution synthesis owing to the soft acid preferring to combine with the soft base and vice versa. We prepared Bu4N1-xLixMnxPb1-xI3 (x = 0.011-0.14) by mechanochemical synthesis. The doping induced a structural phase transition at ∼342 K and much enhancement of ionic conduction above 342 K for all co-doped hybrids regarding Bu4NPbI3 because of the voids around the Mn2+/Li+ ions by doping.

Organic-inorganic haloargentate hybrids of [Me-dabco]Ag2X3 (X = I or Br) with halide ions manipulating the crystal structure, phase transition, and dielectric behavior.

Two haloargentate hybrids, [Me-dabco]Ag2X3 (Me-dabco = 1-methyl-1,4-diazabicyclo-[2.2.2]octan-1-ium, X = I (1) or Br (2)), with the same formula but different structures have been synthesized by a slow evaporation method and characterized by microanalysis, infrared spectroscopy, thermogravimetric, and powder X-ray diffraction techniques. Hybrid 1 consists of completely isolated [Ag4I6]2- clusters, while hybrid 2 exhibits a complicated one-dimensional (1D) chain structure formed by four different configurations of neutral chains and two dissimilar configurations of anionic chains. Hybrid 2 undergoes two reversible order-disorder phase transitions, while hybrid 1 displays one reversible and one irreversible structural phase transition. Both 1 and 2 displayed step-like dielectric anomalies in the vicinity of the phase transition temperature. The corresponding dielectric constants in the high dielectric states are approximately 13 and 6 times higher than those in the low dielectric states for 1 and 2, respectively. Interestingly, the subtle change of halides from I- to Br- significantly affects the aggregated structure of haloargentate, the phase transition, and dielectric behaviors, revealing the typical 'butterfly effect' with the ion radii of halides in these two haloargentate hybrids.

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.

N20D/N116E Combined Mutant Downward Shifted the pH Optimum of Bacillus subtilis NADH Oxidase.

Cofactor regeneration is indispensable to avoid the addition of large quantities of cofactor NADH or NAD+ in oxidation-reduction reactions. Water-forming NADH oxidase (Nox) has attracted substantive attention as it can oxidize cytosolic NADH to NAD+ without concomitant accumulation of by-products. However, its applications have some limitations in some oxidation-reduction processes when its optimum pH is different from its coupled enzymes. In this study, to modify the optimum pH of BsNox, fifteen relevant candidates of site-directed mutations were selected based on surface charge rational design. As predicted, the substitution of this asparagine residue with an aspartic acid residue (N22D) or with a glutamic acid residue (N116E) shifts its pH optimum from 9.0 to 7.0. Subsequently, N20D/N116E combined mutant could not only downshift the pH optimum of BsNox but also significantly increase its specific activity, which was about 2.9-fold at pH 7.0, 2.2-fold at pH 8.0 and 1.2-fold at pH 9.0 that of the wild-type. The double mutant N20D/N116E displays a higher activity within a wide range of pH from 6 to 9, which is wider than the wide type. The usability of the BsNox and its variations for NAD+ regeneration in a neutral environment was demonstrated by coupling with a glutamate dehydrogenase for α-ketoglutaric acid (α-KG) production from L-glutamic acid (L-Glu) at pH 7.0. Employing the variation N20D/N116E as an NAD+ regeneration coenzyme could shorten the process duration; 90% of L-Glu were transformed into α-KG within 40 min vs. 70 min with the wild-type BsNox for NAD+ regeneration. The results obtained in this work suggest the promising properties of the BsNox variation N20D/N116E are competent in NAD+ regeneration applications under a neutral environment.

A {Cu2I3-}∞ chain hybrid with two-step phase transition, switchable dielectrics, thermochromism and piezochromism.

Stimuli-responsive smart materials have applications in a range of technologies. Herein, we present a hybrid (1), built from Me3EtN+ organic cations and {Cu2I3-}∞ inorganic chains with Cu⋯Cu metal⋯metal interactions. The two-step phase transition undergone in 1 on the first heating and the phase transition at a lower temperature show symmetry-broken features, leading to switchable dielectrics; the one at a higher temperature displays isomorphic characteristics. Besides the switchable dielectrics, 1 exhibited other multi-stimuli-responsive functionalities, including thermochromism and piezochromism. Combining temperature-dependent powder and single crystal X-ray diffraction, as well as variable-temperature UV-visible absorption spectrum and EPR spectrum analyses, it is demonstrated that the thermochromism is due to the synergy of anharmonic fluctuations with electron-phonon couplings, and the piezochromism arises from compression inducing a lattice distortion in 1. Our study provides insight into understanding the thermochromic and piezochromic mechanisms of cuprous halide-based hybrids, and paves a pathway for designing new multi-stimuli-responsive hybrid materials.

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.

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.

Efficient Whole-Cell Biotransformation for α-Arbutin Production through the Engineering of Sucrose Phosphorylase Combined with Engineered Cell Modification.

α-Arbutin is extensively used in cosmetic industries. The lack of highly active enzymes and the cytotoxicity of hydroquinone limit the biosynthesis of α-arbutin. In this study, a whole-cell biocatalytic approach based on enzyme engineering and engineered cell modification was identified as effective in enhancing α-arbutin production. First, a sucrose phosphorylase (SPase) mutant with higher enzyme activity was obtained by experimental screening. Next, to avoid the oxidation of hydroquinone, we established an anaerobic process to improve the robustness of the cells by knocking out lytC, sdpC, and skfA in Bacillus subtilis and overcoming the inhibitory effect of a high concentration of hydroquinone. Finally, the engineered strain was used for biotransformation in a 5 L fermenter with batch feeding for 24 h. The final yield of α-arbutin achieved was 129.6 g/L, which may provide a basis for the large-scale industrial production of α-arbutin.

Enhancing erythritol production from crude glycerol in a wild-type Yarrowia lipolytica by metabolic engineering.

Background: Erythritol is a zero-calorie sweetener that is widely used in the food, pharmaceutical, and medical industries. Crude glycerol is the main by-product of biodiesel, and the effective utilization of crude glycerol will help to improve biodiesel viability. Previous studies on the production of erythritol from Y. lipolytica using crude glycerol as a carbon source have focused on optimizing the fermentation process of the mutant Y. lipolytica Wratislavia K1, while metabolic engineering has not been successfully applied. Results: To this end, we engineered the yeast Y. lipolytica to increase the productivity of this strain. Wild strains tolerant to high concentrations of crude glycerol were screened and identified. A series of rational metabolic approaches were employed to improve erythritol production. Among them, the engineered strain Y-04, obtained by tandem overexpression of GUT1 and GUT2, significantly increased glycerol assimilation by 33.3%, which was consistent with the results of RT-qPCR analysis. The effects of tandem overexpression of GUT1, GUT2, TKL1, and TAL1 on erythritol synthesis were also evaluated. The best results were obtained using a mutant that overexpressed GUT1, GUT2, and TKL1 and knocked out EYD1. The final Y-11 strain produced 150 g/l erythritol in a 5-L bioreactor with a yield and productivity of 0.62 g/g and 1.25 g/l/h, respectively. To the best of our knowledge, this is the highest erythritol yield and productivity from crude glycerol ever reported in Y. lipolytica. Conclusion: This work demonstrated that overexpression of GUT1, GUT2, and TKL1 and knockdown of EYD1 could be used to improve crude glycerol utilization and erythritol synthesis in Y. lipolytica. The process parameters such as erythritol yield and productivity were significantly elevated, which is valuable for industrial applications. Crude glycerol, as a carbon source, could efficiently restrict the synthesis of by-products while enhancing the generation of erythritol, compared to glucose. This indicates considerable potential for synthesizing value-added products from crude glycerol by Y. lipolytica.

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.

Acetoin production from lignocellulosic biomass hydrolysates with a modular metabolic engineering system in Bacillus subtilis.

BACKGROUND: Acetoin (AC) is a vital platform chemical widely used in food, pharmaceutical and chemical industries. With increasing concern over non-renewable resources and environmental issues, using low-cost biomass for acetoin production by microbial fermentation is undoubtedly a promising strategy. RESULTS: This work reduces the disadvantages of Bacillus subtilis during fermentation by regulating genes involved in spore formation and autolysis. Then, optimizing intracellular redox homeostasis through Rex protein mitigated the detrimental effects of NADH produced by the glycolytic metabolic pathway on the process of AC production. Subsequently, multiple pathways that compete with AC production are blocked to optimize carbon flux allocation. Finally, the population cell density-induced promoter was used to enhance the AC synthesis pathway. Fermentation was carried out in a 5-L bioreactor using bagasse lignocellulosic hydrolysate, resulting in a final titer of 64.3 g/L, which was 89.5% of the theoretical yield. CONCLUSIONS: The recombinant strain BSMAY-4-PsrfA provides an economical and efficient strategy for large-scale industrial production of acetoin.

[Efficient biosynthesis of D-mannitol by coordinated expression of a two-enzyme cascade].

D-mannitol is widely used in the pharmaceutical and medical industries as an important precursor of antitumor drugs and immune stimulants. However, the cost of the current enzymatic process for D-mannitol synthesis is high, thus not suitable for commercialization. To address this issue, an efficient mannitol dehydrogenase LpGDH used for the conversion and a glucose dehydrogenase BaGDH used for NADH regeneration were screened, respectively. These two enzymes were co-expressed in Escherichia coli BL21(DE3) to construct a two-enzyme cascade catalytic reaction for the efficient synthesis of d-mannitol, with a conversion rate of 59.7% from D-fructose achieved. The regeneration of cofactor NADH was enhanced by increasing the copy number of Bagdh, and a recombinant strain E. coli BL21/pETDuet-Lpmdh-Bagdh-Bagdh was constructed to address the imbalance between cofactor amount and key enzyme expression level in the two-enzyme cascade catalytic reaction. An optimized whole cell transformation process was conducted under 30 ℃, initial pH 6.5, cell mass (OD600) 30, 100 g/L D-fructose substrate and an equivalent molar concentration of glucose. The highest yield of D-mannitol was 81.9 g/L with a molar conversion rate of 81.9% in 5 L fermenter under the optimal conversion conditions. This study provides a green and efficient biotransformation method for future large-scale production of D-mannitol, which is also of great importance for the production of other sugar alcohols.