Efficient biosynthesis of γ-aminobutyric acid by rationally engineering the catalytic pH range of a glutamate decarboxylase from Lactobacillus plantarum / 生物工程学报

γ-aminobutyric acid can be produced by a one-step enzymatic reaction catalyzed by glutamic acid decarboxylase. The reaction system is simple and environmentally friendly. However, the majority of GAD enzymes catalyze the reaction under acidic pH at a relatively narrow range. Thus, inorganic salts are usually needed to maintain the optimal catalytic environment, which adds additional components to the reaction system. In addition, the pH of solution will gradually rise along with the production of γ-aminobutyric acid, which is not conducive for GAD to function continuously. In this study, we cloned the glutamate decarboxylase LpGAD from a Lactobacillus plantarum capable of efficiently producing γ-aminobutyric acid, and rationally engineered the catalytic pH range of LpGAD based on surface charge. A triple point mutant LpGADS24R/D88R/Y309K was obtained from different combinations of 9 point mutations. The enzyme activity at pH 6.0 was 1.68 times of that of the wild type, suggesting the catalytic pH range of the mutant was widened, and the possible mechanism underpinning this increase was discussed through kinetic simulation. Furthermore, we overexpressed the Lpgad and LpgadS24R/D88R/Y309K genes in Corynebacterium glutamicum E01 and optimized the transformation conditions. An optimized whole cell transformation process was conducted under 40 ℃, cell mass (OD600) 20, 100 g/L l-glutamic acid substrate and 100 μmol/L pyridoxal 5-phosphate. The γ-aminobutyric acid titer of the recombinant strain reached 402.8 g/L in a fed-batch reaction carried out in a 5 L fermenter without adjusting pH, which was 1.63 times higher than that of the control. This study expanded the catalytic pH range of and increased the enzyme activity of LpGAD. The improved production efficiency of γ-aminobutyric acid may facilitate its large-scale production.

Abrogation of HnRNP L enhances anti-PD-1 therapy efficacy via diminishing PD-L1 and promoting CD8+ T cell-mediated ferroptosis in castration-resistant prostate cancer

Owing to incurable castration-resistant prostate cancer (CRPC) ultimately developing after treating with androgen deprivation therapy (ADT), it is vital to devise new therapeutic strategies to treat CRPC. Treatments that target programmed cell death protein 1 (PD-1) and programmed death ligand-1 (PD-L1) have been approved for human cancers with clinical benefit. However, many patients, especially prostate cancer, fail to respond to anti-PD-1/PD-L1 treatment, so it is an urgent need to seek a support strategy for improving the traditional PD-1/PD-L1 targeting immunotherapy. In the present study, analyzing the data from our prostate cancer tissue microarray, we found that PD-L1 expression was positively correlated with the expression of heterogeneous nuclear ribonucleoprotein L (HnRNP L). Hence, we further investigated the potential role of HnRNP L on the PD-L1 expression, the sensitivity of cancer cells to T-cell killing and the synergistic effect with anti-PD-1 therapy in CRPC. Indeed, HnRNP L knockdown effectively decreased PD-L1 expression and recovered the sensitivity of cancer cells to T-cell killing in vitro and in vivo, on the contrary, HnRNP L overexpression led to the opposite effect in CRPC cells. In addition, consistent with the previous study, we revealed that ferroptosis played a critical role in T-cell-induced cancer cell death, and HnRNP L promoted the cancer immune escape partly through targeting YY1/PD-L1 axis and inhibiting ferroptosis in CRPC cells. Furthermore, HnRNP L knockdown enhanced antitumor immunity by recruiting infiltrating CD8+ T cells and synergized with anti-PD-1 therapy in CRPC tumors. This study provided biological evidence that HnRNP L knockdown might be a novel therapeutic agent in PD-L1/PD-1 blockade strategy that enhanced anti-tumor immune response in CRPC.

Engineering the C4 pathway of Corynebacterium glutamicum for efficient production of 5-aminolevulinic acid / 生物工程学报

5-aminolevulinic acid (5-ALA) plays an important role in the fields of medicine and agriculture. 5-ALA can be produced by engineered Escherichia coli and Corynebacterium glutamicum. We systematically engineered the C4 metabolic pathway of C. glutamicum to further improve its ability to produce 5-ALA. Firstly, the hemA gene encoding 5-ALA synthase (ALAS) from Rhodobacter capsulatus and Rhodopseudomonas palustris were heterologously expressed in C. glutamicum, respectively. The RphemA gene of R. palustris which showed relatively high enzyme activity was selected. Screening of the optimal ribosome binding site sequence RBS5 significantly increased the activity of RphemA. The ALAS activity of the recombinant strain reached (221.87±3.10) U/mg and 5-ALA production increased by 14.3%. Subsequently, knocking out genes encoding α-ketoglutarate dehydrogenase inhibitor protein (odhI) and succinate dehydrogenase (sdhA) increased the flux of succinyl CoA towards the production of 5-ALA. Moreover, inhibiting the expression of hemB by means of sRNA reduced the degradation of 5-ALA, while overexpressing the cysteine/O-acetylserine transporter eamA increased the output efficiency of intracellular 5-ALA. Shake flask fermentation using the engineered strain C. glutamicum 13032/∆odhI/∆sdhA-sRNAhemB- RBS5RphemA-eamA resulted in a yield of 11.90 g/L, which was 57% higher than that of the original strain. Fed-batch fermentation using the engineered strain in a 5 L fermenter produced 25.05 g/L of 5-ALA within 48 h, which is the highest reported-to-date yield of 5-ALA from glucose.

Rational design of the C-terminal Loop region of leucine dehydrogenase and cascade biosynthesis L-2-aminobutyric acid / 生物工程学报

Leucine dehydrogenase (LDH) is the key rate-limiting enzyme in the production of L-2-aminobutyric acid (L-2-ABA). In this study, we modified the C-terminal Loop region of this enzyme to improve the specific enzyme activity and stability for efficient synthesis of L-2-ABA. Using molecular dynamics simulation of LDH, we analyzed the change of root mean square fluctuation (RMSF), rationally designed the Loop region with greatly fluctuated RMSF, and obtained a mutant EsLDHD2 with a specific enzyme activity 23.2% higher than that of the wild type. Since the rate of the threonine deaminase-catalyzed reaction converting L-threonine into 2-ketobutyrate was so fast, the multi-enzyme cascade catalysis system became unbalanced. Therefore, the LDH and the formate dehydrogenase were double copied in a new construct E. coli BL21/pACYCDuet-RM. Compared with E. coli BL21/pACYCDuet-RO, the molar conversion rate of L-2-ABA increased by 74.6%. The whole cell biotransformation conditions were optimized and the optimal pH, temperature and substrate concentration were 7.5, 35 °C and 80 g/L, respectively. Under these conditions, the molar conversion rate was higher than 99%. Finally, 80 g and 40 g L-threonine were consecutively fed into a 1 L reaction mixture under the optimal conversion conditions, producing 97.9 g L-2-ABA. Thus, this strategy provides a green and efficient synthesis of L-2-ABA, and has great industrial application potential.

Efficient cascade biosynthesis of (S)-2-hydroxybutyric acid / 生物工程学报

2-Hydroxybutyric acid (2-HBA) is an important intermediate for synthesizing biodegradable materials and various medicines. Chemically synthesized racemized 2-HBA requires deracemization to obtain optically pure enantiomers for industrial application. In this study, we designed a cascade biosynthesis system in Escherichia coli BL21 by coexpressing L-threonine deaminase (TD), NAD-dependent L-lactate dehydrogenase (LDH) and formate dehydrogenase (FDH) for production of optically pure (S)-2-HBA from bulk chemical L-threonine (L-Thr). To coordinate the production rate and the consumption rate of the intermediate 2-oxobutyric acid in the multi-enzyme cascade catalytic reactions, we explored promoter engineering to regulate the expression levels of TD and FDH, and developed a recombinant strain P21285FDH-T7V7827 with a tunable system to achieve a coordinated multi-enzyme expression. The recombinant strain P21285FDH-T7V7827 was able to efficiently produce (S)-2-HBA with the highest titer of 143 g/L and a molar yield of 97% achieved within 16 hours. This titer was approximately 1.83 times than that of the highest yield reported to date, showing great potential for industrial application. Our results indicated that constructing a multi-enzyme-coordinated expression system in a single cell significantly contributed to the biosynthesis of hydroxyl acids.

Molecular modification and highly efficient expression of L-asparaginase from Rhizomucor miehei / 生物工程学报

L-asparaginase hydrolyzes L-asparagine to produce L-aspartic acid and ammonia. It is widely distributed in microorganisms, plants and serum of some rodents, and has important applications in the pharmaceutical and food industries. However, the poor thermal stability, low catalytic efficiency and low yield hampered the further application of L-asparaginase. In this paper, rational design and 5' untranslated region (5'UTR) design strategies were used to increase the specific enzyme activity and protein expression of L-asparaginase derived from Rhizomucor miehei (RmAsnase). The results showed that among the six mutants constructed through homology modeling combined with sequence alignment, the specific enzyme activity of the mutant A344E was 1.5 times higher than the wild type. Subsequently, a food-safe strain Bacillus subtilis 168/pMA5-A344E was constructed, and the UTR strategy was used for the construction of recombinant strain B. subtilis 168/pMA5 UTR-A344E. The enzyme activity of B. subtilis 168/pMA5 UTR-A344E was 7.2 times higher than that of B. subtilis 168/pMA5-A344E. The recombinant strain B. subtilis 168/pMA5 UTR-A344E was scaled up in 5 L fermenter, and the final yield of L-asparaginase was 489.1 U/mL, showing great potential for industrial application.

Advances in stress tolerance mechanisms and synthetic biology for the industrial robustness of Corynebacterium glutamicum / 生物工程学报

As a model industrial host and microorganism with the generally regarded as safe (GRAS) status, Corynebacterium glutamicum not only produces amino acids on a large scale in the fermentation industry, but also has the potential to produce various new products. C. glutamicum usually encounters various stresses in the process of producing compounds, which severely affect cell viability and production performance. The development of synthetic biology provides new technical means for improving the robustness of C. glutamicum. In this review, we discuss the tolerance mechanisms of C. glutamicum to various stresses in the fermentation process. At the same time, we highlight new synthetic biology strategies for boosting C. glutamicum robustness, including discovering new stress-resistant elements, modifying transcription factors, and using adaptive evolution strategies to mine stress-resistant functional modules. Finally, prospects of improving the robustness of engineered C. glutamicum strains ware provided, with an emphasis on biosensor, screening and design of transcription factors, and utilizing the multiple regulatory elements.

Synthesis of L-2-aminobutyric acid by leucine dehydrogenase coupling with an NADH regeneration system / 生物工程学报

In this study, Escherichia coli BL21 (DE3) was used as the host to construct 2 recombinant E. coli strains that co-expressed leucine dehydrogenase (LDH, Bacillus cereus)/formate dehydrogenase (FDH, Ancylobacter aquaticus), or leucine dehydrogenase (LDH, Bacillus cereus)/alcohol dehydrogenase (ADH, Rhodococcus), respectively. L-2-aminobutyric acid was then synthesized by L-threonine deaminase (L-TD) with LDH-FDH or LDH-ADH by coupling with two different NADH regeneration systems. LDH-FDH process and LDH-ADH process were optimized and compared with each other. The optimum reaction pH of LDH-FDH process was 7.5, and the optimum reaction temperature was 35 °C. After 28 h, the concentration of L-2-aminobutyric acid was 161.8 g/L with a yield of 97%, when adding L-threonine in batches for controlling 2-ketobutyric acid concentration less than 15 g/L and using 50 g/L ammonium formate, 0.3 g/L NAD+, 10% LDH-FDH crude enzyme solution (V/V) and 7 500 U/L L-TD. The optimum reaction pH of LDH-ADH process was 8.0, and the optimum reaction temperature was 35 °C. After 24 h, the concentration of L-2-aminobutyric acid was 119.6 g/L with a yield of 98%, when adding L-threonine and isopropanol (1.2 times of L-threonine) in batches for controlling 2-ketobutyric acid concentration less than 15 g/L, removing acetone in time and using 0.3 g/L NAD⁺, 10% LDH-ADH crude enzyme solution (V/V) and 7 500 U/L L-TD. The process and results used in this paper provide a reference for the industrialization of L-2-aminobutyric acid.

Effect of key notes of TCA cycle on L-glutamate production / 生物工程学报

Glutamic acid is an important amino acid with wide range of applications and huge market demand. Therefore, by performing transcriptome sequencing and re-sequencing analysis on Corynebacterium glutamicum E01 and high glutamate-producing strain C. glutamicum G01, we identified and selected genes with significant differences in transcription and gene levels in the central metabolic pathway that may have greatly influenced glutamate synthesis and further increased glutamic acid yield. The oxaloacetate node and α-ketoglutarate node play an important role in glutamate synthesis. The oxaloacetate node and α-ketoglutarate node were studied to explore effect on glutamate production. Based on the integrated strain constructed from the above experimental results, the growth rate in a 5-L fermenter was slightly lower than that of the original strain, but the glutamic acid yield after 48 h reached (136.1±5.53) g/L, higher than the original strain (93.53±4.52) g/L, an increase by 45.5%; sugar-acid conversion rate reached 58.9%, an increase of 13.7% compared to 45.2% of the original strain. The application of the above experimental strategy improved the glutamic acid yield and the sugar-acid conversion rate, and provided a theoretical basis for the metabolic engineering of Corynebacterium glutamicum.

Heterologous expression of Streptomyces coelicolor trehalose synthase and whole-cell biocatalyst production of trehalose in Escherichia coli / 生物工程学报

The trehalose synthase (ScTreS) gene from Streptomyces coelicolor was successfully cloned and heterologously expressed in Escherichia coli BL21(DE3). The protein purified by Ni-NTA affinity column showed an apparent molecular weight (MW) of 62.3 kDa analyzed by SDS-PAGE. The optimum temperature of the enzyme was 35 °C and the optimum pH was 7.0; the enzyme was sensitive to acidic conditions. By homologous modeling and sequence alignment, the enzyme was modified by site-directed mutagenesis. The relative activities of the mutant enzymes K246A and A165T were 1.43 and 1.39 times that of the wild type, an increased conversion rate of 14% and 10% respectively. To optimize the synthesis conditions of trehalose, the mutant strain K246A was cultivated in a 5-L fermentor and used for whole-cell transformation. The results showed that with the substrate maltose concentration of 300 g/L at 35 °C and pH 7.0, the highest conversion rate reached 71.3%, and the yield of trehalose was 213.93 g/L. However, when maltose concentration was increased to 700 g/L, the yield of trehalose can reach 465.98 g/L with a conversion rate of 66%.

Contrast-enhanced Ultrasound Guided Percutaneous Puncture Injecting for Hemostatic Control of Active Bleeding of Closed Renal Trauma: An Experimental Study / 中国医学影像学杂志

Purpose Contrast-enhanced ultrasound (CEUS) guided percutaneous puncture injection of hemostatic agents or biological glue is good for the control of closed renal trauma,but there are also some shortcomings.This study aims to explore the effect of CEUS guided percutaneous puncture injection of hemostatic agent and xanthan gum on the control of closed renal trauma hemorrhage.Materials and Methods Sixteen healthy white rabbits were established closed renal trauma models of active bleeding,and were randomly divided into two groups,puncture and laparotomy group.The puncture group was treated with CEUS guided percutaneous injection of snake venom enzyme and biological glue into the active renal hemorrhage.The laparotomy group was treated with traditional surgical suture.The therapeutic effect of two groups was evaluated after operation.The treatment time was recorded,and the renal function was measured every week after the operation.The pathological examination of the wound tissue was performed 1 month after operation.Results Two groups were successful hemostasis.The operation time of puncture group and open group was (179 ± 30) s and (298 ± 30) s,respectively,and the difference was statistically significant (P＜0.05).After operation,the serum creatinine of the puncture group was lower than that of the laparotomy group,and the difference was statistically significant (P＜0.05).The recovery of renal function of the puncture group was faster than that of the laparotomy group,and the renal wound healing was better than that of the laparotomy group.Conclusion The effect of CEUS guided percutaneous puncture injection of snake venom enzyme combined with biological glue in the treatment of closed renal trauma hemorrhage is good,which is a new method worth recommending for easy operation,small damage and quick recovery.

Production of L-citrulline by a recombinant Corynebacterium crenatum SYPA 5-5 whole-cell biocatalyst / 生物工程学报

Arginine deiminase (ADI) was first high-efficient expressed in Corynebacterium crenatum SYPA 5-5. The ADI was purified by Ni-NTA affinity chromatography and SDS-PAGE analysis showed the molecular weight (MW) was 46.8 kDa. The optimal temperature and pH of ADI were 37 ℃ and 6.5 respectively. The Michaelis constant was 12.18 mmol/L and the maximum velocity was 0.36 μmol/(min·mL). Under optimal conditions, 300 g/L of arginine was transformed and the productivity reach 8 g/(L·h). The recombinant strain was cultivated in a 5-L fermentor and used for whole-cell transformation of 300 g/L arginine, under repeated-batch bioconversion, the cumulative production reached 1 900 g/L.

Whole-cell biotransformation for simultaneous synthesis of L-2-aminobutyric acid and D-gluconic acid in recombinant Escherichia coli / 生物工程学报

A whole-cell catalyst using Escherichia coli BL21(DE3) as a host, expressing L- threonine dehydratase from Escherichia coli, and co-expressing leucine dehydrogenase from Bacillus cereus and glucose dehydrogenase from Bacillus subtilis for cofactor regeneration, was constructed and used for one-pot production of L-2-aminobutyric acid (L-ABA) and D- gluconic acid from L-threonine and D-glucose. We used shake-flask culture to study the whole-cell catalytic condition including temperature, pH, proper permeabilization of cells and optimal wet cells amount. Moreover, the whole-cell catalyst was cultured in 5-L fermentor by fed-batch fermentation, and 164 g/L L-threonine and 248 g/L D-glucose were converted to 141.6 g/L L-ABA and 269.4 g/L D-gluconic acid. The whole-cell catalyst is promising to fulfill industrial requirements for L-ABA and D-gluconic acid.

Effects of pH and oxygen supply on production of 2,3-butanediol from biodiesel-derived glycerol by Bacillus amyloliquefaciens / 生物工程学报

Bacillus amyloliquefaciens B10-127 was used to produce 2,3-butanediol (2,3-BD) from residual glycerol obtained from biodiesel synthesis. Important variables for 2,3-BD fermentation, pH and dissolved oxygen, were studied. When pH was maintained constant, the yield of 2,3-BD was inhibited. The highest 2,3-BD yields were achieved by fermentation without any pH control with an optimized initial pH 6.5. Batch fermentative production of 2,3-BD by B. amyloliquefaciens was investigated using various oxygen supply methods by changing agitation speed. Based on the analysis of three kinetic parameters including specific cell growth rate (micro), specific glucose consumption rate (q(s)) and specific 2,3-BD formation rate (q(p)), a three-stage agitation speed control strategy was proposed, aimed at achieving high concentration, high yield and high productivity of 2,3-BD. Maximum concentration of 2,3-BD reached 38.1 g/L, with the productivity of 1.06 g/(L x h), which were 14.8% and 63.1% over the best results from constant agitation speeds. In a pulse fed-batch fermentation, 2,3-BD concentration and productivity were significantly improved to 71.2 g/L and 0.99 g/(L x h), respectively. To our knowledge, these results were the highest for 2,3-BD production from biodiesel-derived glycerol.