Deciphering styrene oxide tolerance mechanisms in Gluconobacter oxydans mutant strain.

Chemical production wastewater contains large amounts of organic solvents (OSs), which pose a significant threat to the environment. In this study, a 10 g·L-1 styrene oxide tolerant strain with broad-spectrum OSs tolerance was obtained via adaptive laboratory evolution. The mechanisms underlying the high OS tolerance of tolerant strain were investigated by integrating physiological, multi-omics, and genetic engineering analyses. Physiological changes are one of the main factors responsible for the high OS tolerance in mutant strains. Moreover, the P-type ATPase GOX_RS04415 and the LysR family transcriptional regulator GOX_RS04700 were also verified as critical genes for styrene oxide tolerance. The tolerance mechanisms of OSs can be used in biocatalytic chassis cell factories to synthesize compounds and degrade environmental pollutants. This study provides new insights into the mechanisms underlying the toxicological response to OS stress and offers potential targets for enhancing the solvent tolerance of G. oxydans.

Design-build-test of recombinant Bacillus subtilis chassis cell by lifespan engineering for robust bioprocesses.

Microbial cell factories utilize renewable raw materials for industrial chemical production, providing a promising path for sustainable development. Bacillus subtilis is widely used in industry for its food safety properties, but challenges remain in the limitations of microbial fermentation. This study proposes a novel strategy based on lifespan engineering to design robust B. subtilis chassis cells to supplement traditional metabolic modification strategies that can alleviate cell autolysis, tolerate toxic substrates, and get a higher mass transfer efficiency. The modified chassis cells could produce high levels of l-glutaminase, and tolerate hydroquinone to produce α-arbutin efficiently. In a 5 L bioreactor, the l-glutaminase enzyme activity of the final strain CRE15TG was increased to 2817.4 ± 21.7 U mL-1, about 1.98-fold compared with that of the wild type. The α-arbutin yield of strain CRE15A was increased to 134.7 g L-1, about 1.34-fold compared with that of the WT. To our knowledge, both of the products in this study performed the highest yields reported so far. The chassis modification strategy described in this study can Improve the utilization efficiency of chassis cells, mitigate the possible adverse effects caused by excessive metabolic modification of engineered strains, and provide a new idea for the future design of microbial cell factories.

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.

N-terminal truncation (N-) and directional proton transfer in an old yellow enzyme enables tunable efficient producing (R)- or (S)-citronellal.

(R)-Citronellal is a valuable molecule as the precursor for the industrial synthesis of (-)-menthol, one of the worldwide best-selling compounds in the flavors and fragrances field. However, its biocatalytic production, even from the optically pure substrate (E)-citral, is inherently limited by the activity of Old Yellow Enzyme (OYE). Herein, we rationally designed a different approach to increase the activity of OYE in biocatalytic production. The activity of OYE from Corynebacterium glutamicum (CgOYE) is increased, as well as superior thermal stability and pH tolerance via truncating the different lengths of regions at N-terminal of CgOYE. Next, we converted the truncation mutant N31-CgOYE, a protein involved in proton transfer for the asymmetric hydrogenation of CC bonds, into highly (R)- and (S)-stereoselective enzymes using only three mutations. The mixture of racemic (E/Z)-citral is reduced into the (R)-citronellal with ee and conversion up to 99 % by the mutant of CgOYE, overcoming the problem of the reduction for the mixtures of (E/Z)-citral in biocatalytic reaction. The present work provides a general and effective strategy for improving the activity of OYE, in which the partially conserved histidine residues provide "tunable gating" for the enantioselectivity for both the (R)- and (S)-isomerases.

Engineering of human tryptophan hydroxylase 2 for efficient synthesis of 5-hydroxytryptophan.

L-Tryptophan hydroxylation catalyzed by tryptophan hydroxylase (TPH) presents a promising method for synthesizing 5-hydroxytryptophan (5-HTP), yet the limited activity of wild-type human TPH2 restricts its application. A high-activity mutant, MT10 (H318E/H323E), was developed through semi-rational active site saturation testing (CAST) of wild-type TPH2, exhibiting a 2.85-fold increase in kcat/Km over the wild type, thus enhancing catalytic efficiency. Two biotransformation systems were developed, including an in vitro one-pot system and a Whole-Cell Catalysis System (WCCS). In the WCCS, MT10 achieved a conversion rate of only 31.5 % within 32 h. In the one-pot reaction, MT10 converted 50 mM L-tryptophan to 44.5 mM 5-HTP within 8 h, achieving an 89 % conversion rate, outperforming the M1 (NΔ143/CΔ26) variant. Molecular dynamics simulations indicated enhanced interactions of MT10 with the substrate, suggesting improved binding affinity and system stability. This study offers an effective approach for the efficient production of 5-HTP.

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.

Novel cytochrome P450s for various hydroxylation of steroids from filamentous fungi.

Hydroxylated steroids are value-added products with diverse biological activities mediated by cytochrome P450 enzymes, however, few has been thoroughly characterized in fungi. This study introduces a rapid identification strategy for filamentous fungi P450 enzymes through transcriptome and bioinformatics analysis. Five novel enzymes (CYP68J5, CYP68L10, CYP68J3, CYP68N1 and CYP68N3) were identified and characterized in Saccharomyces cerevisiae or Aspergillus oryzae. Molecular docking and dynamics simulations were employed to elucidate hydroxylation preferences of CYP68J5 (11α, 7α bihydroxylase) and CYP68N1 (11α hydroxylase). Additionally, redox partners (cytochrome P450 reductase and cytochrome b5) and ABC transporter were co-expressed with CYP68N1 to enhance 11α-OH-androstenedione (11α-OH-4AD) production. The engineered cell factory, co-expressing CPR1 and CYP68N1, achieved a significant increase of 11α-OH-4AD production, reaching 0.845 g·L-1, which increased by 14 times compared to the original strain. This study provides a comprehensive approach for identifying and implementing novel cytochrome P450 enzymes, paving the way for sustainable production of steroidal products.

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.

Microbial production of L-methionine and its precursors using systems metabolic engineering.

L-methionine is an essential amino acid with versatile applications in food, feed, cosmetics and pharmaceuticals. At present, the production of L-methionine mainly relies on chemical synthesis, which conflicts with the concern over serious environmental problems and sustainable development goals. In recent years, microbial production of natural products has been amply rewarded with the emergence and rapid development of system metabolic engineering. However, efficient L-methionine production by microbial fermentation remains a great challenge due to its complicated biosynthetic pathway and strict regulatory mechanism. Additionally, the engineered production of L-methionine precursors, L-homoserine, O-succinyl-L-homoserine (OSH) and O-acetyl-L-homoserine (OAH), has also received widespread attention because they can be catalyzed to L-methionine via a high-efficiently enzymatic reaction in vitro, which is also a promising alternative to chemical route. This review provides a comprehensive overview on the recent advances in the microbial production of L-methionine and its precursors, highlighting the challenges and potential solutions for developing L-methionine microbial cell factories from the perspective of systems metabolic engineering, aiming to offer guidance for future engineering.

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.

[Rational metabolic engineering of Corynebacterium glutamicum for efficient synthesis of L-glutamate].

L-glutamic acid is the world's largest bulk amino acid product that is widely used in the food, pharmaceutical and chemical industries. Using Corynebacterium glutamicum G01 as the starting strain, the fermentation by-product alanine content was firstly reduced by knocking out the gene encoding alanine aminotransferase (alaT), a major by-product related to alanine synthesis. Secondly, since the α-ketoglutarate node carbon flow plays an important role in glutamate synthesis, the ribosome-binding site (RBS) sequence optimization was used to reduce the activity of α-ketoglutarate dehydrogenase and enhance the glutamate anabolic flow. The endogenous conversion of α-ketoglutarate to glutamate was also enhanced by screening different glutamate dehydrogenase. Subsequently, the glutamate transporter was rationally desgined to improve the glutamate efflux capacity. Finally, the fermentation conditions of the strain constructed using the above strategy were optimized in 5 L fermenters by a gradient temperature increase combined with a batch replenishment strategy. The glutamic acid production reached (135.33±4.68) g/L, which was 41.2% higher than that of the original strain (96.53±2.32) g/L. The yield was 55.8%, which was 11.6% higher than that of the original strain (44.2%). The combined strategy improved the titer and the yield of glutamic acid, which provides a reference for the metabolic modification of glutamic acid producing strains.

[Rational design of polyphosphate kinase dual-substrate channel cavity for efficient production of glutathione by cell free catalysis].

ATP is an important cofactor involved in many biocatalytic reactions that require energy input. Polyphosphate kinases (PPK) can provide energy for ATP-consuming reactions due to their cheap and readily available substrate polyphosphate. We selected ChPPK from Cytophaga hutchinsonii for substrate profiling and tolerance analysis. By molecular docking and site-directed mutagenesis, we rationally engineered the dual-substrate channel cavity of polyphosphate kinase to improve the catalytic activity of PPK. Compared with the wild type, the relative enzyme activity of the screened mutant ChPPKK81H-K103V increased by 326.7%. Meanwhile, the double mutation expanded the substrate utilization range and tolerance of ChPPK, and improved its heat and alkali resistance. Subsequently, we coupled the glutathione bifunctional enzyme GshAB and ChPPKK81H-K103V based on this ATP regeneration system, and glutathione was produced by cell-free catalysis upon disruption of cells. This system produced (25.4±1.9) mmol/L glutathione in 6 h upon addition of 5 mmol/L ATP. Compared with the system before mutation, glutathione production was increased by 41.9%. After optimizing the buffer, bacterial mass and feeding time of this system, (45.2±1.8) mmol/L glutathione was produced in 6 h and the conversion rate of the substrate l-cysteine was 90.4%. Increasing the ability of ChPPK enzyme to produce ATP can effectively enhance the conversion rate of substrate and reduce the catalytic cost, achieving high yield, high conversion rate and high economic value for glutathione production by cell-free catalysis. This study provides a green and efficient ATP regeneration system that may further power the ATP-consuming biocatalytic reaction platform.

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.

L-lysine production by systems metabolic engineering of an NADPH auto-regulated Corynebacterium glutamicum.

Here, the systems metabolic engineering of L-lysine-overproducing Corynebacterium glutamicum is described to create a highly efficient microorganism producer. The key chromosomal mutations associated with L-lysine synthesis were identified based on whole-genome sequencing. The carbon flux was subsequently redirected into the L-lysine synthesis pathway and increased the availability of energy and product transport systems required for L-lysine synthesis. In addition, a promoter library sensitive to intracellular L-lysine concentration was constructed and applied to regulate the NADPH pool dynamically. In the fed-batch fermentation experiment, the L-lysine titer of the final engineered strain was 223.4 ± 6.5 g/L. This study is the first to improve L-lysine production by enhancing ATP supply and NADPH self-regulation to improve the intracellular environment.

The flavohaemoprotein hmp maintains redox homeostasis in response to reactive oxygen and nitrogen species in Corynebacterium glutamicum.

BACKGROUND: During the production of L-arginine through high dissolved oxygen and nitrogen supply fermentation, the industrial workhorse Corynebacterium glutamicum is exposed to oxidative stress. This generates reactive oxygen species (ROS) and reactive nitrogen species (RNS), which are harmful to the bacteria. To address the issue and to maintain redox homeostasis during fermentation, the flavohaemoprotein (Hmp) was employed. RESULTS: The results showed that the overexpression of Hmp led to a decrease in ROS and RNS content by 9.4% and 22.7%, respectively, and improved the survivability of strains. When the strains were treated with H2O2 and NaNO2, the RT-qPCR analysis indicated an up-regulation of ammonium absorption and transporter genes amtB and glnD. Conversely, the deletion of hmp gives rise to the up-regulation of eight oxidative stress-related genes. These findings suggested that hmp is associated with oxidative stress and intracellular nitrogen metabolism genes. Finally, we released the inhibitory effect of ArnR on hmp. The Cc-ΔarnR-hmp strain produced 48.4 g/L L-arginine during batch-feeding fermentation, 34.3% higher than the original strain. CONCLUSIONS: This report revealed the influence of dissolved oxygen and nitrogen concentration on reactive species of Corynebacterium glutamicum and the role of the Hmp in coping with oxidative stress. The Hmp first demonstrates related to redox homeostasis and nitrite metabolism, providing a feasible strategy for improving the robustness of strains.

A high-throughput dual system to screen polyphosphate kinase mutants for efficient ATP regeneration in L-theanine biocatalysis.

ATP, an important cofactor, is involved in many biocatalytic reactions that require energy. Polyphosphate kinases (PPK) can provide energy for ATP-consuming reactions due to their cheap and readily available substrate polyphosphate. We determined the catalytic properties of PPK from different sources and found that PPK from Cytophaga hutchinsonii (ChPPK) had the best catalytic activity for the substrates ADP and polyP6. An extracellular-intracellular dual system was constructed to high-throughput screen for better catalytic activity of ChPPK mutants. Finally, the specific activity of ChPPKD82N-K103E mutant was increased by 4.3 times. Therefore, we focused on the production of L-theanine catalyzed by GMAS as a model of ATP regeneration. Supplying 150 mM ATP, GMAS enzyme could produce 16.8 ± 1.3 g/L L-theanine from 100 mM glutamate. When 5 mM ATP and 5 U/mL ChPPKD82N-K103E were added, the yield of L-theanine was 16.6 ± 0.79 g/L with the conversion rate of 95.6 ± 4.5% at 4 h. Subsequently, this system was scaled up to 200 mM and 400 mM glutamate, resulting in the yields of L-theanine for 32.3 ± 1.6 g/L and 62.7 ± 1.1 g/L, with the conversion rate of 92.8 ± 4.6% and 90.1 ± 1.6%, respectively. In addition, we also constructed an efficient ATP regeneration system from glutamate to glutamine, and 13.8 ± 0.2 g/L glutamine was obtained with the conversion rate of 94.4 ± 1.4% in 4 h after adding 6 U/ mL GS enzyme and 5 U/ mL ChPPKD82N-K103E, which further laid the foundation from glutamine to L-theanine catalyzed by GGT enzyme. This proved that giving the reaction an efficient ATP supply driven by the mutant enzyme enhanced the conversion rate of substrate to product and maximized the substrate value. This is a positively combination of high yield, high conversion rate and high economic value of enzyme catalysis. The mutant enzyme will further power the ATP-consuming biocatalytic reaction platform sustainably.

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.