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1,4-butanediol is an important intermediate widely used in chemical, agricultural, and pharmaceutical industries. This study constructed a new short path for the production of 1,4-butanediol with glucose as the substrate by combining enzyme engineering and metabolic engineering. Firstly, a novel path catalyzed by α-ketoglutarate decarboxylase (SucA), carboxylate reductase (Car), and alcohol dehydrogenase (YqhD) was designed by database mining, and the de novo synthesis of 1,4-butanediol was achieved after introduction of the path into Escherichia coli W3110 (K-12) chassis cells. To further improve the synthesis efficiency of this path, we deleted the genes encoding lactate dehydrogenase A (LdhA) and pyruvate formate lyase B (PflB) to block the metabolic bypass. Furthermore, the expression of citrate synthase (GltA R163L ) was up-regulated to increase the α-ketoglutarate metabolic flux. In addition, we improved the synthesis of the key cofactor NADPH and up-regulated the expression of sucA , car , and yqhD by substituting with strong promoters to increase the efficiency of supplying precursors to 1,4-butanediol synthesis. Eventually, the recombinant strain produced up to 770 mg/L of 1,4-butanediol within 48 h in a shake flask, and 4.22 g/L of 1,4-butanediol within 60 h in a 5 L fermenter with a yield of 12.46 mg/g glucose. Compared with the previously reported method, the novel path designed in this study for the de novo synthesis of 1,4-butanediol does not need acetyl coenzyme A and avoids the byproduct acetate or the addition of ammonia. Therefore, the outcome is expected to provide a new idea for the metabolic engineering of microbial chassis for the production of 1,4-butanediol and its high-value derivatives.

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.

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 Lp GAD from a Lactobacillus plantarum capable of efficiently producing γ-aminobutyric acid, and rationally engineered the catalytic pH range of Lp GAD based on surface charge. A triple point mutant Lp GAD S24R/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 Lpgad S24R/D88R/Y309K genes in Corynebacterium glutamicum E01 and optimized the transformation conditions. An optimized whole cell transformation process was conducted under 40 ℃, cell mass ( OD 600 ) 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 Lp GAD. The improved production efficiency of γ-aminobutyric acid may facilitate its large-scale production.

Biliverdin is an important cellular antioxidant. Traditionally, biliverdin is produced by chemical oxidation of bilirubin, which is a complex process and the final product is of low purity. Here we report an efficient, green and safe process for biotechnological production of biliverdin. A heme oxygenase (HO) gene from Clostridium tetani was screened, and a recombinant strain Escherichia coli BL21/pETDuet- hoCt with the ability of transforming heme into biliverdin was constructed. A biliverdin yield of 32.9 mg/L from 100 mg/L substrate was achieved under pH 7.0 and 35 ℃. In order to improve the supply of reducing power, an NADPH regeneration system using glutamate dehydrogenase (GdhA) was constructed, resulting in a recombinant strain E . coli BL21/pETDuet- gdhAEc - hoCt which was capable of producing 71.5 mg/L biliverdin. Moreover, through introduction of a membrane surface display system, a recombinant strain E . coli BL21/pETDuet- gdhAEc - blc / hoCt was constructed to shorten the transformation time, and the production of biliverdin was further increased to 76.3 mg/L, this is the highest titer of biosynthesized biliverdin reported to date, and the research may thus facilitate the green production of biliverdin.

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.

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.

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.

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.

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.

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.

2-O-α-D-glu-copyranosyl-sn-glycerol is a high value-added product with prospective application in food, cosmetics, health products and pharmaceutical industries. However, industrial scale of 2-O-α-D-glu-copyranosyl-sn-glycerol has not yet been applied in China, and there are few related reports on 2-O-α-D-glu-copyranosyl-sn-glycerol synthesis. The purpose of this experiment is to develop a method for catalyzing the synthesis of food-grade 2-O-α-D-glu-copyranosyl-sn-glycerol using whole cells of "Generally Recognized as Safe" (GRAS) recombinant Bacillus subtilis. In our work, a recombinant B. subtilis 168/pMA5-gtfA that heterologously expressing Leuconostoc mesenteroides sucrose phosphorylase was constructed and used as a whole-cell catalyst to synthesize 2-O-α-D-glu-copyranosyl-sn-glycerol. Optimizing the culture temperature, time and whole cell transformation conditions has increased the yield of 2-O-α-D-glu-copyranosyl-sn-glycerol. The results showed that 1.43 U/mL of sucrose phosphorylase was achieved in B. subtilis 168/pMA5-gtfA after culturing for 20 h at 30 °C in fermentation medium. The highest conversion rate reached 75.1%, and the yield of 2-O-α-D-glu-copyranosyl-sn-glycerol was 189.3 g/L with an average transformation rate of 15.6 mmol/(L·h) after 48 hours whole-cell transformation with the sucrose concentration of 1 mol/L and the glycerol concentration of 2.5 mol/L at 30 °C, OD₆₀₀ 40 and pH 7.0. This is the highest yield of 2-O-α-D-glu-copyranosyl-sn-glycerol synthesized catalytically by recombinant B. subtilis that was ever reported, and this study provides the theoretical and experimental basis for the industrial production and application of 2-O-α-D-glucopyranosyl-sn-glycerol.

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.

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%.

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.

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.

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.