Biosynthetic L-tert-leucine using Escherichia coli co-expressing a novel NADH-dependent leucine dehydrogenase and a formate dehydrogenase

BackgroundL-tert-Leucine has been widely used in pharmaceutical, chemical, and other industries as a vital chiral intermediate. Compared with chemical methods, enzymatic methods to produce L-tert-leucine have unparalleled advantages. Previously, we found a novel leucine dehydrogenase from the halophilic thermophile Laceyella sacchari (LsLeuDH) that showed good thermostability and great potential for the synthesis of L-tert-leucine in the preliminary study. Hence, we manage to use the LsLeuDH coupling with a formate dehydrogenase from Candida boidinii (CbFDH) in the biosynthesis of L-tert-leucine through reductive amination in the present study.ResultThe double-plasmid recombinant strain exhibited higher conversion than the single-plasmid recombinant strain when resting cells cultivated in shake flask for 22 h were used. Under the optimized conditions, the double-plasmid recombinant E. coli BL21 (pETDute-FDH-LDH, pACYCDute-FDH) transformed 1 mol·L^-1 trimethylpyruvate (TMP) completely into L-tert-leucine with greater than 99.9% ee within 8 h.ConclusionsThe LsLeuDH showed great ability to biosynthesize L-tert-leucine. In addition, it provided a new option for the biosynthesis of L-tert-leucine.How to citeWang L, Zhu W, Gao Z, et al. Biosynthetic L-tert-leucine using Escherichia coli co-expressing a novel NADH-dependent leucine dehydrogenase and a formate dehydrogenase. Electron J Biotechnol 2020;47. https://doi.org/10.1016/j.ejbt.2020.07.001     

Escherichia coli expressing endoglucanase gene from Thai higher termite bacteria for enzymatic and microbial hydrolysis of cellulosic materials

BackgroundEndoglucanase plays a major role in initiating cellulose hydrolysis. Various wild-type strains were searched to produce this enzyme, but mostly low extracellular enzyme activities were obtained. To improve extracellular enzyme production for potential industrial applications, the endoglucanase gene of Bacillus subtilis M015, isolated from Thai higher termite, was expressed in a periplasmic-leaky Escherichia coli. Then, the crude recombinant endoglucanase (EglS) along with a commercial cellulase (Cel) was used for hydrolyzing celluloses and microbial hydrolysis using whole bacterial cells.ResultsE. coli Glu5 expressing endoglucanase at high levels was successfully constructed. It produced EglS (55 kDa) with extracellular activity of 18.56 U/mg total protein at optimal hydrolytic conditions (pH 4.8 and 50°C). EglS was highly stable (over 80% activity retained) at 40–50°C after 100 h. The addition of EglS significantly improved the initial sugar production rates of Cel on the hydrolysis of carboxymethyl cellulose (CMC), microcrystalline cellulose, and corncob about 5.2-, 1.7-, and 4.0-folds, respectively, compared to those with Cel alone. E. coli Glu5 could secrete EglS with high activity in the presence of glucose (1% w/v) and Tween 80 (5% w/v) with low glucose consumption. Microbial hydrolysis of CMC using E. coli Glu5 yielded 26 mg reducing sugar/g CMC at pH 7.0 and 37°C after 48 h.ConclusionsThe recombinant endoglucanase activity improved by 17 times compared with that of the native strain and could greatly enhance the enzymatic hydrolysis of all studied celluloses when combined with a commercial cellulase.     

Construction and application of a novel genetically engineered Aspergillus oryzae for expressing proteases

BackgroundWe aimed to test the possibility of improving polypeptide production from soybean meal fermentation by engineered Aspergillus oryzae strains. Four different protease genes were cloned and transformed into wild-type A. oryzae, and the engineered A. oryzae strains were then used for soybean meal fermentation.ResultsThe results showed different degrees of improvement in the protease activity of the four transformants when compared with wild-type A. oryzae. A major improvement in the polypeptide yield was achieved when these strains were used in soybean meal fermentation. The polypeptide conversion rate of one of the four transformants, A. oryzae pep, reached 35.9%, which was approximately twofold higher than that exhibited by wild-type A. oryzae. Amino acid content analysis showed that the essential amino acid content and amino acid composition of the fermentation product significantly improved when engineered A. oryzae strains were used for soybean meal fermentation.ConclusionsThese findings suggest that cloning of microbial protease genes with good physicochemical properties and expressing them in an ideal host such as A. oryzae is a novel strategy to enhance the value of soybean meal.     

Metabolic engineering strategies for caffeic acid production in Escherichia coli

Caffeic acid (CA; 3,4-dihydroxycinnamic acid) is an aromatic compound obtained by the phenylpropanoid pathway. This natural product has antioxidant, antitumor, antiviral, and anti-inflammatory activities. It is also a precursor of CA phenethyl ester (CAPE), a compound with potential as an antidiabetic and liver-protective agent. CA can be found at low concentrations in plant tissues, and hence, its purification is difficult and expensive. Knowledge regarding the pathways, enzymes, and genes involved in CA biosynthesis has paved the way for enabling the design and construction of microbial strains with the capacity of synthesizing this metabolite. In this review, metabolic engineering strategies for the generation of Escherichia coli strains for the biotechnological production of CA are presented and discussed.How to cite: Hernández-Chávez G, Martinez A, Gosset G. Metabolic engineering strategies for caffeic acid production in Escherichia coli. Electron J Biotechnol 2019;38. https://doi.org/10.1016/j.ejbt.2018.12.004.     

大肠杆菌氨基酸转运蛋白的研究进展

总结了大肠杆菌氨基酸的原初主动转运蛋白、次级主动转运蛋白和易化扩散载体在其种类、功能、结构和转运机制方面的研究进展.详细介绍了氨基酸与势能离子相伴的同向共转运和反向共转运,并提出了大肠杆菌氨基酸转运研究中存在问题及展望.     

Construction of an RNAi vector for knockdown of GM-ACS genes in the cotyledonary nodes of soybean

BackgroundEthylene plays an important role in the regulation of floral organ development in soybean, and 1-aminocyclopropane-1-carboxylate synthase (ACS) is a rate-limiting enzyme for ethylene biosynthesis. However, whether ACS also regulates floral organ differentiation in soybean remains unknown. To address this, we constructed an RNAi vector to inhibit ACS expression in cotyledonary nodes. Linear DNA cassettes of RNAi-ACS obtained by PCR were used to transform soybean cotyledonary nodes.ResultsIn total, 131 of 139 transiently transformed plants acquired herbicide resistance and displayed GUS activities in the new buds. In comparison to untransformed seedling controls, a greater number of flower buds were differentiated at the cotyledonary node; GM-ACS1 mRNA expression levels and ethylene emission in the transformed buds were reduced.ConclusionThese results indicate that the cotyledonary node transient transformation system may be suitable for stable transformation and that the inhibition of ACS expression may be an effective strategy for promoting floral organ differentiation in soybean.     

Improvement of polyhydroxybutyrate production by deletion of csrA in Escherichia coli

BackgroundPoly-3-hydroxybutyrate (PHB) can be efficiently produced in recombinant Escherichia coli by the overexpression of an operon (NphaCAB) encoding PHB synthetase. Strain improvement is considered to be one of critical factors to lower the production cost of PHB in recombinant system. In this study, one of key regulators that affect the cell growth and PHB content was confirmed and analyzed.ResultS17-3, a mutant E. coli strain derived from S17-1, was found to be able to achieve high cell density when expressing NphaCAB with the plasmid pBhya-CAB. Whole genome sequencing of S17-3 revealed genetic alternations on the upstream regions of csrA, encoding a global regulator cross-talking between stress response, catabolite repression and other metabolic activities. Deletion of csrA or expression of mutant csrA resulted in improved cell density and PHB content.ConclusionThe impact of gene deletion of csrA was determined, dysfunction of the regulators improved the cell density of recombinant E. coli and PHB production, however, the detail mechanism needs to be further clarified.How to cite: Wu H, Li S, Ji M, et al. Improvement of polyhydroxybutyrate production by deletion of csrA in Escherichia coli. Electron J Biotechnol 2020;46. https://doi.org/10.1016/j.ejbt.2020.04.005.     

Dielectrophoretic microfluidic device for the continuous sorting of Escherichia coli from blood cells

Microfluidic diagnostic devices promise faster disease identification by purifying and concentrating low-abundance analytes from a flowing sample. The diagnosis of sepsis, a whole body inflammatory response often caused by microbial infections of the blood, is a model system for pursuing the advantages of microfluidic devices over traditional diagnostic protocols. Traditional sepsis diagnoses require large blood samples and several days to culture and identify the low concentration microbial agent. During these long delays while culturing, the physician has little or no actionable information to treat this acute illness. We designed a microfluidic chip using dielectrophoresis to sort and concentrate the target microbe from a flowing blood sample. This design was optimized using the applicable electrokinetic and hydrodynamic theories. We quantify the sorting efficiency of this device using growth-based assays which show 30% of injected microbes are recovered viable, consistent with the electroporation of target cells by the dielectrophoretic cell sorters. Finally, the results illustrate the device is capable of a five-fold larger microbe concentration in the target analyte stream compared to the waste stream at a continuous sample flow rate of 35 μl∕h.     

Vif基因体外表达载体构建及其在无细胞系统中的表达

随着-omics（组学）时代的到来,无细胞蛋白质合成系统以具有快速、方便、易于高通量等优点,正在被广泛地研究和应用。本文选取HIV病毒感染因子Vif作为目标蛋白,构建了适宜体外表达的Vif表达载体pIVEX2.4c-Vif,并将其在大肠杆菌无细胞蛋白质合成系统中进行表达,为下一步进行高通量药物筛选奠定了一定的基础。     

西藏牦牛大肠埃希氏菌黏附素及胞外蛋白酶的测定

本文通过对牦牛致病性大肠埃希氏菌(西藏-9903)和大肠埃希氏菌标准菌种(C83907)黏附素的测定,红细胞凝集谱测定试验表明,西藏-9903只能凝集家兔红细胞,血凝谱窄,凝集价为29,C83907能凝集家兔、鸡、鸭、猪红细胞,血凝谱广,凝集价分别为29、211、28、28;通过对西藏-9903菌株和C83907进行菌毛小肠上皮细胞刷状缘黏附试验,结果表明,每一个乳兔小肠上皮刷状缘细胞至少能吸附7个菌细胞,二者均能较强或强黏附于刷状缘细胞上。胞外蛋白酶活性试验检测结果为西藏-9903菌株和C83907检测均为阳性,说明黏附素及胞外蛋白酶是牦牛大肠埃希氏菌重要的毒力因子,此研究为该病的防制提供一定科学依据。     

Bifurcation analysis of a competitive system with general toxic production and delayed toxic effects

Population interaction may release poisonous chemicals to inhibit other species’ growth in the ecosystem, especially for the competitive populations. The negative effect of toxic chemical substances may not display immediately and appear with time lag during the species’ growth. In this work, we investigate a competitive system with the delayed toxic effects of the chemicals from species interaction. Theoretical results obtained in this work help us reveal the delayed toxic factors on species’ growth. We first consider the existence and the stability of the equilibria. The influence of delay terms on the positive steady state is validated. The delayed toxic effects here will contribute to the oscillation for the concentration of species when the value of time delay passes through a critical point. Besides, the stability of periodic solutions from the Hopf bifurcation and the direction of the Hopf bifurcation are also determined. Finally, several numerical examples are provided to validate the theoretical conclusions.     

Improvement of hydrogen yield of ethanol-producing Escherichia coli recombinants in acidic conditions

BackgroundAn effective single culture with high glycerol consumption and hydrogen and ethanol coproduction yield is still in demand. A locally isolated glycerol-consuming Escherichia coli SS1 was found to produce lower hydrogen levels under optimized ethanol production conditions. Molecular approach was proposed to improve the hydrogen yield of E. coli SS1 while maintaining the ethanol yield, particularly in acidic conditions. Therefore, the effect of an additional copy of the native hydrogenase gene hycE and recombinant clostridial hydrogenase gene hydA on hydrogen production by E. coli SS1 at low pH was investigated.ResultsRecombinant E. coli with an additional copy of hycE or clostridial hydA was used for fermentation using 10 g/L (108.7 mmol/L) of glycerol with an initial pH of 5.8. The recombinant E. coli with hycE and recombinant E. coli with hydA showed 41% and 20% higher hydrogen yield than wild-type SS1 (0.46 ± 0.01 mol/mol glycerol), respectively. The ethanol yield of recombinant E. coli with hycE (0.50 ± 0.02 mol/mol glycerol) was approximately 30% lower than that of wild-type SS1, whereas the ethanol yield of recombinant E. coli with hydA (0.68 ± 0.09 mol/mol glycerol) was comparable to that of wild-type SS1.ConclusionsInsertion of either hycE or hydA can improve the hydrogen yield with an initial pH of 5.8. The recombinant E. coli with hydA could retain ethanol yield despite high hydrogen production, suggesting that clostridial hydA has an advantage over the hycE gene in hydrogen and ethanol coproduction under acidic conditions. This study could serve as a useful guidance for the future development of an effective strain coproducing hydrogen and ethanol.     

Escherichia coli GutM4 produces 2,5-diketopiperazines and inhibits human pathogens in vitro

BackgroundIt has been a very common practice to use probiotics or their metabolites as alternative antimicrobial strategies for the treatment and prevention of infections as rampant and indiscriminate use of antibiotics causes the development of antibiotic-resistant pathogens. The objective of this study was to select a potential antimicrobial probiotic strain of Escherichia coli from the human gastrointestinal tract and investigate the production of diketopiperazines that contribute to the antimicrobial activity.ResultsE. coli GutM4 was isolated from the feces of a healthy adult. E. coli GutM4 showed significant antagonistic activity against 10 indicator pathogens, and this activity was no less than that of the reference strain E. coli Nissle 1917 against eight of the indicator pathogens. Moreover, E. coli GutM4 produced antagonistic substances containing trypsin-targeted peptide bonds because the inhibitory effects of E. coli GutM4 supernatant significantly decreased upon treatment with trypsin. Consistent with the antagonistic activity and peptide compounds of E. coli GutM4, 14 2,5-diketopiperazines were isolated from the fermented broth of E. coli GutM4, including 12 cyclo(Pro-Phe), 3 cyclo(Pro-Tyr), and 5 cyclo(4-hydroxyl-Pro-Leu), which are reported to have antipathogenic activity.ConclusionE. coli GutM4 produces 2,5-diketopiperazines that are partly involved in antagonistic action against human pathogens in vitro.     

大肠杆菌的mRNA的翻译效率决定于Shine-Dalgarno的局部结构

在大肠杆菌中,多胺对mRNA翻译的影响程度决定于Shine-Dalgarno (SD)序列局部结构。与信使RNA其它区域松散配对的SD序列的局部结构受多胺的扰动较大,其局部解链的概率也较大,因而SD序列结合到核糖体RNA上并启动蛋白质翻译的概率也较大,相反,则启动mRNA翻译的概率就会较小。基于此,建立一个简单数学模型来描述这一现象,即蛋白质的表达水平是与由多胺刺激而引起mRNA的SD序列局部暴露的概率相关的。结果显示,模型与实验结果相符的很好。     

Co-production of hydrogen and ethanol by Escherichia coli SS1 and its recombinant

BackgroundThe development of a potential single culture that can co-produce hydrogen and ethanol is beneficial for industrial application. Strain improvement via molecular approach was proposed on hydrogen and ethanol co-producing bacterium, Escherichia coli SS1. Thus, the effect of additional copy of native hydrogenase gene hybC on hydrogen and ethanol co-production by E. coli SS1 was investigated.ResultsBoth E. coli SS1 and the recombinant hybC were subjected to fermentation using 10 g/L of glycerol at initial pH 7.5. Recombinant hybC had about 2-fold higher cell growth, 5.2-fold higher glycerol consumption rate and 3-fold higher ethanol productivity in comparison to wild-type SS1. Nevertheless, wild-type SS1 reported hydrogen yield of 0.57 mol/mol glycerol and ethanol yield of 0.88 mol/mol glycerol, which were 4- and 1.4-fold higher in comparison to recombinant hybC. Glucose fermentation was also conducted for comparison study. The performance of wild-type SS1 and recombinant hybC showed relatively similar results during glucose fermentation. Additional copy of hybC gene could manipulate the glycerol metabolic pathway of E. coli SS1 under slightly alkaline condition.ConclusionsHybC could improve glycerol consumption rate and ethanol productivity of E. coli despite lower hydrogen and ethanol yields. Higher glycerol consumption rate of recombinant hybC could be an advantage for bioconversion of glycerol into biofuels. This study could serve as a useful guidance for dissecting the role of hydrogenase in glycerol metabolism and future development of effective strain for biofuels production.     

Media development and process parameter optimization using statistical experimental designs for the production of nonribosomal peptides in Escherichia coli

BackgroundNonribosomal peptide synthases (NRPS) can synthesize functionally diverse bioactive peptides by incorporating nonproteinogenic amino acids, offering a rich source of new drug leads. The bacterium Escherichia coli is a well-characterized production host and a promising candidate for the synthesis of nonribosomal peptides, but only limited bioprocess engineering has been reported for such molecules. We therefore developed a medium and optimized process parameters using the design of experiments (DoE) approach.ResultsWe found that glycerol is not suitable as a carbon source for rhabdopeptide production, at least for the NRPS used for this study. Alternative carbon sources from the tricarboxylic acid cycle achieved much higher yields. DoE was used to optimize the pH and temperature in a stirred-tank reactor, revealing that optimal growth and optimal production required substantially different conditions.ConclusionsWe developed a chemically defined adapted M9 medium matching the performance of complex medium (lysogeny broth) in terms of product concentration. The maximum yield in the reactor under optimized conditions was 126 mg L^-1, representing a 31-fold increase compared to the first shaking-flask experiments with M9 medium and glycerol as the carbon source. Conditions that promoted cell growth tended to inhibit NRPS productivity. The challenge was therefore to find a compromise between these factors as the basis for further process development.How to cite: Oestreich AM, Suli LI, Gerlach D. et al. Media development and process parameter optimization using statistical experimental designs for the production of nonribosomal peptides in Escherichia coli. Electron J Biotechnol 2021;52. https://doi.org/10.1016/j.ejbt.2021.05.001     

Expression,purification and thermal stability evaluation of an engineered amaranth protein expressed in Escherichia coli

BackgroundThe acidic subunit of amarantin (AAC)—the predominant amaranth seed storage protein—has functional potential and its third variable region (VR) has been modified with antihypertensive peptides to improve this potential. Here, we modified the C-terminal in the fourth VR of AAC by inserting four VY antihypertensive peptides. This modified protein (AACM.4) was expressed in Escherichia coli. In addition, we also recombinantly expressed other derivatives of the amarantin protein. These include: unmodified amarantin acidic subunit (AAC); amarantin acidic subunit modified at the third VR with four VY peptides (AACM.3); and amarantin acidic subunit doubly modified, in the third VR with four VY peptides and in the fourth VR with the RIPP peptide (AACM.3.4).ResultsE. coli BL21-CodonPlus (DE3)-RIL was the most favorable strain for the expression of proteins. After 6 h of induction, it showed the best recombinant protein titer. The AAC and AACM.4 were obtained at higher titers (0.56 g/L) while proteins modified in the third VR showed lower titers: 0.44 g/L and 0.33 g/L for AACM.3 and AACM.3.4, respectively. As these AAC variants were mostly expressed in an insoluble form, we applied a refolding protocol. This made it possible to obtain all proteins in soluble form. Modification of the VR 4 improves the thermal stability of amarantin acidic subunit; AAC manifested melting temperature (T_m) at 34°C and AACM.4 at 37.2°C. The AACM.3 and AACM.3.4 did not show transition curves.ConclusionsModifications to the third VR affect the thermal stability of amarantin acidic subunit.