Co-ordinated combination of Embden-Meyerhof-Parnas pathway and pentose phosphate pathway in Escherichia coli to promote L-tryptophan production

In this study,phosphoenolpyruvate and erythrose-4-phosphate are efficiently supplied by collaborative design of Embden-Meyerhof-Parnas(EMP)pathway and pentose phosphate(PP)pathway in Escherichia coli,thus increasing the L-tryptophan production.Firstly,the effects of disrupting EMP pathway on L-tryptophan production were studied,and the results indicated that the strain with deletion of phosphofructokinase A(i.e.,E.coli JW-5ΔpfkA)produced 23.4±2.1 g/L of L-tryptophan production.However,deletion of phosphofructokinase A and glucosephosphate isomerase is not conducive to glucose consumption and cell growth,especially deletion of glucosephosphate isomerase.Next,the carbon flux in PP pathway was enhanced by introduction of the desensitized glucose-6-phosphate dehydrogenase(zwf)and 6-phosphogluconate dehydrogenase(gnd)and thus increasing the L-tryptophan production(i.e.,26.5±3.2 g/L vs.21.7±1.3 g/L)without obviously changing the cell growth(i.e.,0.41 h^(-1) vs.0.44 h^(-1))as compared with the original strain JW-5.Finally,the effects of co-modifying EMP pathway and PP pathway on L-tryptophan production were investigated.It was found that the strain with deletion of phosphofructokinase A as well as introduction of the desensitized zwf and gnd(i.e.,E.coli JW-5 zwf243 gnd361ΔpfkA)produced 31.9±2.7 g/L of L-tryptophan,which was 47.0%higher than that of strain JW-5.In addition,the glucose consumption rate of strain JW-5 zwf243 gnd361ΔpfkA was obviously increased despite of the bad cell growth as compared with strain JW-5.The results of this study have important reference value for the following application of metabolic engineering to improve aromatic amino acids producing strains.

β-半乳糖苷酶和阿拉伯糖异构酶共表达一步法催化乳糖到塔格糖

【目的】构建一株以廉价原料乳糖为底物合成塔格糖的重组菌株,实现一步法高效生物合成稀有糖——塔格糖。【方法】从Escherichia coli K-12基因组中,PCR扩增出阿拉伯糖异构酶araA和β-半乳糖苷酶lacZ基因,以SD-AS为连接子,利用pET28a-1载体串联表达于Escherichia coli BL21(DE3),获得重组菌E.coli BL21/pET28a-araA-lacZ,对重组菌全细胞催化合成塔格糖的条件进行了工艺优化与放大研究。【结果】araA和lacZ基因在E.coli BL21中同时高效表达,在最优条件(pH 8.0、温度50℃、5 mmol/L Mn^(2+)、添加0.5 mol/L硼酸和0.1%SDS)下,E.coli BL21/pET28a-araA-lacZ全细胞转化100 g/L乳糖,合成塔格糖最高产量达24.03±2.03 g/L,乳糖到塔格糖的摩尔转化率为45.67%,随着底物乳糖浓度的提高,塔格糖产量呈不同程度的提高,当投加500 g/L底物乳糖时,全细胞合成塔格糖产量最高达83.81±1.38 g/L。【结论】通过2个关键靶酶的编码基因araA和lacZ在E.coli BL21细胞中进行共表达,实现了以重组菌全细胞为催化剂转化廉价底物乳糖,一步法高效合成稀有糖塔格糖,该研究为生物法制备低能量的功能性稀有糖奠定了较好的研究基础。

Hepatoprotective ability of tetramethylpyrazine produced by Bacillus amyloliquefaciens

Tetramethylpyrazine(TTMP),an important aroma compound,was produced by Bacillus amyloliquefaciens XJB-104 with distillers’grains as raw material.The yield of TTMP under optimized fermentation procedure was 3176.52 mg/L.TTMP in the fermentation broth was purified and the purity(>99%)was measured by gas chromatography-mass spectrometry.Furthermore,the hepatoprotective activity of TTMP in ethanol-water system was evaluated by biochemical indicators of liver injury,parameters of antioxidant defense system and inflammatory response,and liver histopathological assessment in mice.Ethanol treatment increased serum levels of alanine aminotransferase,aspartate aminotransferase,alkaline phos-phatase and lactate dehydrogenase,suggesting liver damage.Both high and low dose of TTMP significantly decreased levels of these indicators.Furthermore,the reduction in the activities or concentrations of superoxide dismutase,catalase,reduced glutathione and malondialdehyde in liver tissue caused by ethanol was significantly alleviated by TTMP.Increased inflammatory cytokines including transcription factor,tumor necrosis factor,interleukin-1beta,interleukin,macrophage chemoattractant protein,inducible nitric oxide synthase and cyclooxygenase were also suppressed by TTMP treatment.It is concluded that TTMP in ethanol-water system has potential liver-protective activity,especially when ethanol is consumed at low doses for short periods of time.

Biosynthesis, property comparison, and hemocompatibility of bacterial and haloarchaeal poly(3-hydroxybutyrate-co-3-hydroxyvalerate)

Polyhydroxyalkanoates(PHAs) are a class of natural biopolyesters accumulated intracellularly by many microorganisms. These polymers have attracted particular attention as green plastic in biomedical and industrial applications due to their good biodegradability and biocompatibility. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate)(PHBV) is one of the most common members of PHAs. However, there is no report comparing the properties of PHBV from different groups of producers, e.g.,bacteria and haloarchaea. In this study, two types of PHBV copolymers were synthesized in Halogranum amylolyticum and Ralstonia eutropha, respectively, by feeding different carbon sources. They possessed a similar concentration of3 HV monomers(21 mol%) and were named PHBV-H(produced by H. amylolyticum) and PHBV-B(produced by R. eutropha) based on their source. Interestingly, they exhibited different behaviors especially in thermal stability, melting temperature, crystallinity percentage, and mechanical properties. Furthermore, the films of PHBV-Hand PHBV-B possessed different surface properties, such as surface roughness, wettability, and surface free energy.The value of hemolysis on the PHBV-H film was lower in comparison with the PHBV-B film, although both values were within the limit of 5 % permissible for biomaterials.Notably, few inactivated platelets adhered to the surface of the PHBV-H film, whereas numerous activated platelets were seen on film PHBV-B. These results indicated that PHBV-H was a better potential component of blood-contact biomaterials than PHBV-B. Our study clearly revealed that the properties of PHAs are source dependent and haloarchaeal species provide a new opportunity for the production of desired PHAs.

Cascade biocatalysis for production of enantiopure(S)-2-hydroxybutyric acid using recombinant Escherichia coli with a tunable multi-enzyme-coordinate expression system

Racemize 2-hydroxybutyric acid is usually synthesized by organic methods and needs additional deracemization to obtain optically pure enantiomers for industrial application.Here we present a cascade biocatalysis system in Escherichia coli BL21 which employed L-threonine deaminase(TD),NAD-dependent L-lactate dehydrogenase(LDH)and alcohol dehydrogenase(ADH)for producing optically pure(S)-2-hydroxybutyric acid((S)-2-HBA)from bulk chemical L-threonine.To solve the mismatch in the conversion rate and the consumption rate of intermediate 2-oxobutyric acid(2-OBA)formed in the multi-enzyme catalysis reaction,ribosome binding site regulation strategy was explored to control TD expression levels,achieving an eightfold alteration in the conversion rate of 2-OBA.With the optimized activity ratio of the three enzymes and using ADH for NADH regeneration,the recombinant strain ADH-r53 showed increased production of(S)-2-HBA with the highest titer of 129 g/L and molar yield of 93%within 24 h,which is approximately 1.65 times that of the highest yield reported so far.Moreover,(S)-2-HBA could easily be purified by distillation,making it have great potential for industrial application.Additionally,our results indicated that constructing a tunable multi-enzyme-coordinate expression system in single cell had great significance in biocatalysis of hydroxyl acids.

Characterization of Bacillus subtilis Ab03 for efficient ammonia nitrogen removal

An efficient ammonia nitrogen degrading bacterial strain was isolated from a fish and shrimp pond and identified as Bacillus subtilis(Ab03).Firstly,the strain was continuously domesticated in ammonium solution to improve its nitrogen removal capacity.The performance of the strain in terms of nitrogen removal efficiency under different culture conditions was then examined.Finally,the nitrogen removal process and related strain mechanisms were analyzed by nitrogen balance.The results showed the strain Ab03 could remove 91.67% of NH_(4)^(+)-N at 300 mg/L under the conditions of glucose as the single carbon source,C/N of 15,pH of 7.5,the temperature of 35℃ and dissolved oxygen of 7-8 mg/L.It was also found that under conditions where ammonia nitrogen was the only nitrogen source,strain Ab03 could also undergo aerobic denitrification for simultaneous conversion,with a final gas conversion rate of 74.81%.

Comparative transcriptome analysis reveals metabolic regulation of prodigiosin in Serratia marcescens

Prodigiosin is a secondary metabolite mainly produced at 30°C in Serratia marcescens,but it can hardly be synthetized at 37°C or higher.In this study,we provide insight into the metabolic regulation of prodigiosin synthesis in response to temperature through transcriptome sequencing.The analysis of the function of differentially expressed genes suggested that temperature resulted in significant alteration of the metabolic pathways between 30 and 37°C.Specifically,30°C favored transcriptional expression of the pig gene cluster.At the same time,the carbon flux was redistributed to pathways of pyruvate,proline,serine,especially homoserine,cystathionine,homocysteine,methionine,and s-adenosylmethionine metabolism,all involved in prodigiosin biosynthesis,and was finally increased towards the prodigiosin synthesis pathway in S.marcescens at 30°C.Interestingly,results further confirmed increased transcriptional level of five regulators(LuxS,RpoS,Hfq,EepR,CRP),and decreased content of hexS through qPCR.Finally,successful co-overexpression of mmuM and metK,related to homocysteine,methionine,and s-adenosylmethionine metabolism,in the chromosome of JNB5-1(JNB5-1/MK)resulted in increased prodigiosin titer up to 7.57 g/L in JNB5-1/MK at 30°C,which was 41.2%higher than that in JNB5-1.Our transcriptome analysis provides further insight into the strain’s response to temperature changes at the transcription level,which is of great significance for improving the production of prodigiosin.