Revealing the Mechanisms of Enhanced ß-Farnesene Production in Yarrowia lipolytica through Metabolomics Analysis.

ß-Farnesene is an advanced molecule with promising applications in agriculture, the cosmetics industry, pharmaceuticals, and bioenergy. To supplement the shortcomings of rational design in the development of high-producing ß-farnesene strains, a Metabolic Pathway Design-Fermentation Test-Metabolomic Analysis-Target Mining experimental cycle was designed. In this study, by over-adding 20 different amino acids/nucleobases to induce fluctuations in the production of ß-farnesene, the changes in intracellular metabolites in the ß-farnesene titer-increased group were analyzed using non-targeted metabolomics. Differential metabolites that were detected in each experimental group were selected, and their metabolic pathways were located. Based on these differential metabolites, targeted strain gene editing and culture medium optimization were performed. The overexpression of the coenzyme A synthesis-related gene pantothenate kinase (PanK) and the addition of four mixed water-soluble vitamins in the culture medium increased the ß-farnesene titer in the shake flask to 1054.8 mg/L, a 48.5% increase from the initial strain. In the subsequent fed-batch fermentation, the ß-farnesene titer further reached 24.6 g/L. This work demonstrates the tremendous application value of metabolomics analysis for the development of industrial recombinant strains and the optimization of fermentation conditions.

Metabolic Engineering for Effective Synthesis of 2-Hydroxyadipate.

Adipic acid is an important monomer in the synthesis of nylon-6,6. In recent years, the biosynthesis of adipic acid has received more and more attention. The pathway with l-lysine as a precursor has potential for adipic acid synthesis, and 2-hydroxyadipate is a key intermediate metabolite in this pathway. In this Letter, the biosynthesis pathway of 2-hydroxyadipate was constructed in Escherichia coli. Through enhancement of precursor synthesis and cofactors regulation, 7.11 g/L of 2-hydroxyadipate was produced in the 5 L bioreactor, which verified the scale-up potential of 2-hydroxyadipate production. Furthermore, 11.1 g/L of 2-hydroxyadipate was produced in the 5 L bioreactor on the basis of potential optimization strategies via transcriptome analysis. This is the first time for the biosynthesis of 2-hydroxyadipate. The results lay a solid foundation for the biosynthesis of adipic acid and the production of bionylon.

A Highly Sensitive Model Based on Graph Neural Networks for Enzyme Key Catalytic Residue Prediction.

Determining the catalytic site of enzymes is a great help for understanding the relationship between protein sequence, structure, and function, which provides the basis and targets for designing, modifying, and enhancing enzyme activity. The unique local spatial configuration bound to the substrate at the active center of the enzyme determines the catalytic ability of enzymes and plays an important role in the catalytic site prediction. As a suitable tool, the graph neural network can better understand and identify the residue sites with unique local spatial configurations due to its remarkable ability to characterize the three-dimensional structural features of proteins. Consequently, a novel model for predicting enzyme catalytic sites has been developed, which incorporates a uniquely designed adaptive edge-gated graph attention neural network (AEGAN). This model is capable of effectively handling sequential and structural characteristics of proteins at various levels, and the extracted features enable an accurate description of the local spatial configuration of the enzyme active site by sampling the local space around candidate residues and special design of amino acid physical and chemical properties. To evaluate its performance, the model was compared with existing catalytic site prediction models using different benchmark datasets and achieved the best results on each benchmark dataset. The model exhibited a sensitivity of 0.9659, accuracy of 0.9226, and area under the precision-recall curve (AUPRC) of 0.9241 on the independent test set constructed for evaluation. Furthermore, the F1-score of this model is nearly four times higher than that of the best-performing similar model in previous studies. This research can serve as a valuable tool to help researchers understand protein sequence-structure-function relationships while facilitating the characterization of novel enzymes of unknown function.

Synergistic analysis of performance, microbial community, and metabolism in aerobic granular sludge under polyacrylonitrile microplastics stress.

Aerobic granular sludge (AGS) has proved to be a promising biotechnology for microplastics wastewater treatment. However, polyacrylonitrile microplastics (PAN MPs), the most widely used plastic in textile materials, have not been investigated. Therefore, the effect of the neglected PAN MPs on AGS at different concentrations (1, 10, and 100 mg/L) was evaluated. The results indicated that PAN MPs with 1 and 10 mg/L concentrations had no obvious effect on granular stability and nutrient removal performance, but greatly promoted the secretion of EPS. Remarkably, the granule structure was severely damaged under 100 mg/L PAN MPs. Moreover, microbial community analysis showed that phylum Proteobacteria played a dominant role in resistance to PAN MPs. Metabolic analysis further revealed that genes related to denitrification pathway (nasA, nirK, nirS and norB) and membrane transport were significantly inhibited under PAN MPs stress. This study may provide additional information on the treatment of microplastics wastewater using AGS.

Enhancing precursor supply and modulating metabolism to achieve high-level production of ß-farnesene in Yarrowia lipolytica.

ß-Farnesene is a sesquiterpene commonly found in essential oils of plants, with applications spanning from agricultural pest control and biofuels to industrial chemicals. The use of renewable substrates in microbial cell factories offers a sustainable approach to ß-farnesene biosynthesis. In this study, malic enzyme from Mucor circinelloides was examined for NADPH regeneration, concomitant with the augmentation of cytosolic acetyl-CoA supply by expressing ATP-citrate lyase from Mus musculus and manipulating the citrate pathway via AMP deaminase and isocitrate dehydrogenase. Carbon flux was modulated through the elimination of native 6-phosphofructokinase, while the incorporation of an exogenous non-oxidative glycolysis pathway served to bridge the pentose phosphate pathway with the mevalonate pathway. The resulting orthogonal precursor supply pathway facilitated ß-farnesene production, reaching 810 mg/L in shake-flask fermentation. Employing optimal fermentation conditions and feeding strategy, a titer of 28.9 g/L of ß-farnesene was attained in a 2 L bioreactor.

De novo biosynthesis of α-aminoadipate via multi-strategy metabolic engineering in Escherichia coli.

As a non-protein amino acid, α-aminoadipate is used in the fields of medicine, chemical engineering, food science, and others. For example, α-aminoadipate is an important precursor for the production of ß-lactam antibiotics. Currently, the synthesis of α-aminoadipate depends on chemical catalysis that has the disadvantages of high cost, low yield, and serious pollution. In this study, we construct a biosynthesis pathway of α-aminoadipate in Escherichia coli using lysine as a precursor. In addition, we regulate the cell metabolism to improve the titer of α-aminoadipate via multi-strategy metabolic engineering. First, a novel synthetic pathway was constructed to realize de novo synthesis of α-aminoadipate with titers of 82 mg/L. Second, the key enzymes involved in enhancing precursor synthesis were overexpressed and the CO2 fixation process was introduced, and these led to 80% and 34% increases in the α-aminoadipate concentration, reaching 147 and 110 mg/L, respectively. Third, cofactor regulation was used to maintain the coupling balance of material and energy, with the intracellular α-aminoadipate concentration reaching 140 mg/L. Fourth, the weakening of the synthesis of acetic acid was used to strengthen the synthesis of α-aminoadipate, and this resulted in the enhancement of the α-aminoadipate concentration by 2.2 times, reaching 263 mg/L. Finally, combination optimization was used to promote the production of α-aminoadipate. The titers of α-aminoadipate reached 368 mg/L (strain EcN11#) and 415 mg/L (strain EcN11##), which was 3.5 and 4 times higher than that of the parent strain. With these efforts, 1.54 g/L of α-aminoadipate was produced under fed-batch conditions by strain EcN11#. This study is the first to present the effective biosynthesis of α-aminoadipate in E. coli using multi-strategy metabolic engineering.

Prediction of Plasticizer Property Based on an Improved Genetic Algorithm.

Different plasticizers have obvious differences in plasticizing properties. As one of the important indicators for evaluating plasticization performance, the substitution factor (SF) has great significance for product cost accounting. In this research, a genetic algorithm with "variable mutation probability" was developed to screen the key molecular descriptors of plasticizers that are highly correlated with the SF, and a SF prediction model was established based on these filtered molecular descriptors. The results show that the improved genetic algorithm greatly improved the prediction accuracy in different regression models. The coefficient of determination (R2) for the test set and the cross-validation both reached 0.92, which is at least 0.15 higher than the R2 of the unimproved genetic algorithm. From the results of the selected descriptors, most of the descriptors focused on describing the branching of the molecule, which is consistent with the view that the branching chain plays an important role in the plasticization process. As the first study to establish the relationship between plasticizer SF and plasticizer molecular structure, this work provides a basis for subsequent plasticizer performance and evaluation system modeling.

Improved energy efficiency in microbial fuel cells by bioethanol and electricity co-generation.

BACKGROUND: Microbial electricity production has received considerable attention from researchers due to its environmental friendliness and low price. The increase in the number of intracellular electrons in a microbial fuel cell (MFC) helps to improve the MFC performance. RESULTS: In this study, we accumulated excess electrons intracellularly by knocking out the gene related to intracellular electron consumption in Saccharomyces cerevisiae, and the elevated intracellular electron pool positively influenced the performances of MFCs in terms of electricity production, while helping to increase ethanol production and achieve ethanol and electricity co-production, which in turn improved the utilization of substrates. The final knockout strain reached a maximum ethanol yield of 7.71 g/L and a maximum power density of 240 mW/m2 in the MFC, which was 12 times higher than that of the control bacteria, with a 17.3% increase in energy utilization. CONCLUSIONS: The knockdown of intracellular electron-consuming genes reported here allowed the accumulation of excess electrons in cells, and the elevated intracellular electron pool positively influenced the electrical production performance of the MFC. Furthermore, by knocking out the intracellular metabolic pathway, the yield of ethanol could be increased, and co-production of ethanol and electricity could be achieved. Thus, the MFC improved the utilization of the substrate.

Cross-linking of carbonic anhydrase and formate dehydrogenase based on amino acid specific recognition: Conversion of carbon dioxide to formic acid.

Inspired by the cascades performed in vivo, the assembly of multiple enzymes in vitro has strongly moved into the focus of researchers in the field of biocatalysis. In this study, a new, mild and accurate enzyme cross-linking method is revealed. Microbial transglutaminase (MTG) acts as a "cross-linking medium" by identifying the amide group of the glutamine and the primary amine group of lysine in the artificial peptide tags specifically to form an iso-peptide bond. Here, carbonic anhydrase (CA) and formate dehydrogenase (FDH) with different peptide tags that can be recognized by MTG were linked together to obtain different proportions of cross-linked enzymes for efficient conversion of greenhouse gas carbon dioxide to formic acid. After cross-linking, we obtained "one-to-one" and "one-to-more" cross-linked enzyme aggregates. There is a minor residual loss of the two enzymes, the remaining enzyme activity of CA is more than 93%, and the remaining enzyme activity of FDH is more than 84%. In particular, the overall catalytic efficiency of the cross-linked enzyme is increased by 5.8 times compared with free enzymes and the thermal stability of FDH at different temperatures is improved. The applied strategy demonstrates the potential application of MTG in multi-enzyme assembly and synthetic biology.

Quantification of alcohols, diols and glycerol in fermentation with an instantaneous derivatization using trichloroacetyl isocyanante via liquid chromatography-massspectrometry.

A sensitive LC-MS/MS method was established to quantify diols and glycerol in fermentation broth using trichloroacetyl isocyanate as instantaneous derivatization reagent for monitoring the production of 1,3-propanediol and 2,3-butanediol from the biodiesel biorefinery process. Due to the derivatization reaction was very quickly at room temperature, only 1 min was needed for the reaction process. In addition, both extraction of analytes and evaporation of water were not employed in the analytical procedure. Furthermore, the isotope of chlorine was beneficial for understanding of the secondary mass spectrum and avoiding false positive results. Therefore, much more accurate results of diols and glycerol concentration in fermentation could be obtained even at very low levels for the evaluation of microbial metabolism pathway modification.

Simultaneous acetone-butanol-ethanol fermentation, gas stripping, and full-cell-catalyzed esterification for effective production of butyl oleate.

In this study, aiming to improve the economic feasibility of acetone-butanol-ethanol (ABE) fermentation process, generate valuable products and extend the product chain, esterification catalyzed by Candida sp. 99-125 cells was hybrid with the ABE fermentation-gas-stripping integration system. The gas-stripping condensate that contained concentrated ABE products was directly used for esterification without the participation of toxic organic solvents. Full-cell catalysis temperature and the cell dosage rate on oleate production were evaluated and optimized in the esterification process. Under the optimized conditions (35 °C, 8% of cells), ~ 68% of butyl oleate and ~ 12% of ethyl oleate were obtained after 4 h of esterification. The Candida sp. 99-125 cells were able to be reused for at least four cycles. The novel cascade process showed environmental benefits, which also showed promising in improving the economic feasibility of the conventional ABE fermentation process.

Bio-plasticizer production by hybrid acetone-butanol-ethanol fermentation with full cell catalysis of Candida sp. 99-125.

Hybrid process that integrated fermentation, pervaporation and esterification was established aiming to improve the economic feasibility of the conventional acetone-butanol-ethanol (ABE) fermentation process. Candida sp 99-125 cells were used as full-cell catalyst. The feasibility of batch and fed-batch esterification using the ABE permeate of pervaporation (ranging from 286.9 g/L to 402.9 g/L) as substrate were compared. Valuable butyl oleate was produced along with ethyl oleate. For the batch esterification, due to severe inhibition of substrate to lipase, the yield of butyl oleate and ethyl oleate were only 24.9% and 3.3%, respectively. In contrast, 75% and 11.8% of butyl oleate and ethyl oleate were obtained, respectively, at the end of the fed-batch esterification. The novel integration process provides a promising strategy for in situ upgrading ABE products.

Environmentally-friendly strategy for separation of 1,3-propanediol using biocatalytic conversion.

Glycerol waste from the biodiesel production can be used as a carbon source in the production of 1,3-propanediol (1,3-PD) through microbial fermentation. However, downstream processing is a major bottleneck that restricts its biological production. Here, we investigated an environmentally-friendly method to enzymatically separate 1,3-PD. The transformation of 1,3-PD to an ester was achieved by exploiting the esterification reaction with fatty acids under lipase catalysis. The reaction efficiency was optimized using different poly-alcohols that were existed in the fermentation broth reacted with a fatty acid. Whereas the 1,3-PD conversion reached 62%, only a 0.06% and 0.08% conversion was reached for 2,3-butanediol and glycerol, illustrating the former's more efficient separation. The recovery efficiency of 1,3-PD was 96%. Finally, 1,3-PD was obtained by lipase-directed ester hydrolysis. Taken together, the bio-catalyzed separation process presented here is a novel and promising method for recovering 1,3-PD.

Cofactor engineering for more efficient production of chemicals and biofuels.

Cofactors are involved in numerous intracellular reactions and critically influence redox balance and cellular metabolism. Cofactor engineering can support and promote the biocatalysis process, even help driving thermodynamically unfavorable reactions forwards. To achieve efficient production of chemicals and biofuels, cofactor engineering strategies such as altering cofactor supply or modifying reactants' cofactor preference have been developed to maintain redox balance. This review focuses primarily on the effects of cofactor engineering on carbon and energy metabolism. Coupling carbon metabolism with cofactor engineering can promote large-scale production, and even offer possibilities for producing new products or converting new materials.

Enzymatic phosphorylation of mannose by glucomannokinase from Mycobacterium phlei using inorganic polyphosphate.

Mannose-6-phosphate is an important phosphor-sugar, which is involved in many physiological functions and it is used to treat many diseases. Its production is however expensive since it requires costly substrate ATP as phosphorylation agent. This study has focused upon the direct synthesis of M6P by glucomannokinase using inorganic polyphosphate without involvement of ATP. The gene cloned for glucomannokinase has been sequenced from Mycobacterium phlei and it is transformed into Escherichia coli for expression. After purification involving affinity chromatography, a band of 30kDa corresponding to the enzyme has been isolated from induced crude supernatant. A total amount of 0.69mg/ml of enzyme has been successively obtained and the purity exceeds 90%. The kinetic assay studies show that this enzyme has more affinity towards polyphosphate and glucose than ATP and mannose respectively. The KM values of the enzyme for glucose, mannose, ATP and hexametaphosphate derived from experiments are 9.5, 203.7, 4.6, 1.7µM, respectively. The enzyme has shown a maximum production of mannose-6-phosphate at optimized conditions of pH 8.5, 25°C, poly(P)/mannose ratio 3:1 and in the presence of bivalent ion Mg2+. The results reveal that the glucomannokinase from Mycobacterium phlei suitable for further production of mannose-6-phosphate.

Genipin Cross-Linked Glucose Oxidase and Catalase Multi-enzyme for Gluconic Acid Synthesis.

In this work, glucose oxidase (GOD) and catalase (CAT) were used simultaneously to produce gluconic acid from glucose. In order to reduce the distance between the two enzymes, and therefore improve efficiency, GOD and CAT were cross-linked together using genipin. Improvements in gluconic acid production were due to quick removal of harmful intermediate hydrogen peroxide by CAT. GOD activity was significantly affected by the proportion of CAT in the system, with GOD activity in the cross-linked multi-enzyme (CLME) being 10 times higher than that in an un-cross-linked GOD/CAT mixture. The glucose conversion rate after 15 h using 15 % glucose was also 10 % higher using the CLME than was measured using a GOD/CAT mixture.

Immobilization of Yarrowia lipolytica lipase Ylip2 for the biocatalytic synthesis of phytosterol ester in a water activity controlled reactor.

In this work, phytosterol ester was synthesized using Yarrowia lipolytica lipase Ylip2 that had been immobilized on inorganic support in a solvent-free system and reacted in a computer-aided water activity controlled bioreactor. The immobilization of Ylip2 on celite led to a remarkable increase in the phytosterol conversion compared to that of free lipase. An investigation of the reaction conditions were oleic acid as the fatty acid variety, 10,000U/g substrate, and a temperature of 50°C for phytosterol ester synthesis. Controlling of the water activity at a set point was accomplished by the introduction of dry air through the reaction medium at a digital feedback controlled flow rate. For the esterification of phytosterol ester, a low (15%) water activity resulted in a considerable improvement in phytosterol conversion (91.1%) as well as a decreased reaction time (78h). Furthermore, Ylip2 lipase immobilized on celite retained 90% esterification activity for the synthesis of phytosterol oleate after reused 8 cycles, while free lipase was only viable for 5 batches with 90% esterification activity remained. Finally, the phytosterol oleate space time yield increased from 1.65g/L/h with free lipase to 2.53g/L/h with immobilized lipase. These results illustrate that the immobilized Yarrowia lipolytica lipase Ylip2 in a water activity controlled reactor has great potential for the application in phytosterol esters synthesis.

Immobilizing Yarrowia lipolytica Lipase Lip2 via Improvement of Microspheres by Gelatin Modification.

The purpose of this study was to investigate the feasibility of immobilizing Yarrowia lipolytica lipase lip2 on epoxy microspheres with or without gelatin modifications. The activity of lipase immobilized on gelatin-modified supports was twofold higher than those immobilized on native supports. There was no significant difference in the Michaelis-Menten constant (K M ) between the two immobilized lipases. However, lipase immobilized on gelatin modified supports showed an approximately fourfold higher V max than lipase immobilized on native supports. Lipase immobilization on the gelatin-modified support exhibited a significantly improved operational stability in an esterification system. After it was reused for a total of 35 batches, the ester conversion of lipase immobilized on gelatin-modified and native microspheres was 83 and 60 %, respectively. Furthermore, the immobilized lipase could be stored at 4 °C for 12 months without any loss of activity.

Synergistic effects of amine and protein modified epoxy-support on immobilized lipase activity.

We have developed an improved and effective method to immobilize Yarrowia lipolytica lipase Lip2 (YLIP2) on an epoxy poly-(glycidylmethacrylate-triallyisocyanurate-ethyleneglycoldimethacrylate) (PGMA-TAIC-EGDMA) support structure with or without amine or/and protein modifications. Our results show that there is an increase in the activity of the immobilized lipase on n-butylamine (BA) modified support (420U/g support) and the biocompatible gelatin modified support (600U/g support) when compared to the support without modification (240U/g support). To further study the influences of BA and gelatin modification on the activity of the immobilized lipase, gelatin and BA were concurrently used to decorate the support structure. Lipase immobilized on 2% BA/gelatin (1:1) modified support obtained the highest activity (1180U/g support), which was five-fold higher than that on a native support structure. These results suggest that the activity of a support-immobilized lipase depends on the support surface properties and a moderate support surface micro-environment was crucial for elevated activity. Collectively, these data show that a combined gelatin and BA modification regulates the support surface more suitable for immobilizing YLIP2.

Solvothermal synthesis of functional magnetic nanoparticles for biocatalyst.

A facile and simple one-step solvothermal method has been developed to synthesize polyethyleneimine (PEI)-modified magnetic nanoparticles. Characterization of morphology, surface charges, crystal structure, and magnetic property confirmed the efficiency of this facile synthesis route. Lipase immobilized on the PEI-modified magnetic nanoparticles was used to synthesize vitamin A palmitate from vitamin A acetate and palmitic acid. The reuse of immobilized lipase can be extended to eight times by removing water during esterification with a conversion rate above 80 % for 12 h.