Synergetic engineering of Escherichia coli for efficient production of L-tyrosine

L-Tyrosine,an aromatic non-essential amino acid,is the raw material for many important chemical products,including levodopa,resveratrol,and hydroxytyrosol.It is widely used in the food,drug,and chemical industries.There are many studies on the synthesis of L-tyrosine by microorganisms,however,the low titer of L-tyrosine limited the industrial large-scale production.In order to enhance L-tyrosine production in Escherichia coli,the expression of key enzymes in the shikimate pathway was up-or down-regulated.The L-tyrosine transport system and the acetic acid biosynthesis pathway were modified to further enhance L-tyrosine production.In addition,the phosphoketolase pathway was introduced in combination with cofactor engineering to redirect carbon flux to the shikimate pathway.Finally,after adaptive laboratory evolution to low pH an optimal strain was obtained.The strain can produce 92.5 g/L of L-tyrosine in a 5-L fermenter in 62 h,with a yield of 0.266 g/g glucose.

Food‑grade expression of multicopper oxidase with improved capability in degrading biogenic amines

Biogenic amines(BAs)are potential amine hazards that are detected in fermented foods and alcoholic beverages.Excessive intake of BAs may lead to allergic symptoms such as difculty in breathing,nausea,and vomiting.Degradation of BAs by multicopper oxidase(MCO)is a promising method as it has little efect on the fermentation process,food nutrition,and favor.However,the application of MCO in food industry was restricted due to its poor catalytic properties and low productivity.In this work,food-grade expression of the Bacillus amyloliquefaciens MCO(MCOB)and its three mutants were successfully constructed in Lactococcus lactis NZ3900.The expression level of MCOB in L.lactis NZ3900 was dramatically enhanced by optimizing the cultivation conditions,and the highest expression level reached 4488.1 U/L.This was the highest expression level of food-graded MCO reported so far,to our knowledge.Interestingly,the optimal reaction pH of MCOB expressed in L.lactis NZ3900 switched to 4.5,it would be more suitable for degrading BAs in food as the pH value of most fermented foods was found to be 4.5.Moreover,MCOB expressed in L.lactis NZ3900 was quite stable(with more than 80%residual activity)in the pH range of 4.0–5.5,the catalytic rate constant(kcat)and specifc activity of MCOBLS were all dramatically increased compared with that of MCOB expressed in Escherichia coli.Using histamine as the substrate,the degradation of BAs within 24 h by MCOB expressed in L.lactis NZ3900 was 69.7%higher than that expressed in E.coli.The results demonstrated the potential applications of MCOB in food industry for reduction of biogenic amines.

Engineering Saccharomyces cerevisiae for enhanced(–)-α-bisabolol production

(–)-α-Bisabolol is naturally occurring in many plants and has great potential in health products and pharma-ceuticals.However,the current extraction method from natural plants is unsustainable and cannot fulfil the increasing requirement.This study aimed to develop a sustainable strategy to enhance the biosynthesis of(–)-α-bisabolol by metabolic engineering.By introducing the heterologous gene MrBBS and weakening the competitive pathway gene ERG9,a de novo(–)-α-bisabolol biosynthesis strain was constructed that could produce 221.96 mg/L(–)-α-bisabolol.Two key genes for(–)-α-bisabolol biosynthesis,ERG20 and MrBBS,were fused by a flexible linker(GGGS)3 under the GAL7 promoter control,and the titer was increased by 2.9-fold.Optimization of the mevalonic acid pathway and multi-copy integration further increased(–)-α-bisabolol production.To promote product efflux,overexpression of PDR15 led to an increase in extracellular production.Combined with the optimal strategy,(–)-α-bisabolol production in a 5 L bioreactor reached 7.02 g/L,which is the highest titer reported in yeast to date.This work provides a reference for the efficient production of(–)-α-bisabolol in yeast.

Efficient stereoselective hydroxylation of deoxycholic acid by the robust whole-cell cytochrome P450 CYP107D1 biocatalyst

Deoxycholic acid(DCA)has been authorized by the Federal Drug Agency for cosmetic reduction of redundant submental fat.The hydroxylated product(6β-OH DCA)was developed to improve the solubility and pharmaceutic properties of DCA for further applications.Herein,a combinatorial catalytic strategy was applied to construct a powerful Cytochrome P450 biocatalyst(CYP107D1,OleP)to convert DCA to 6β-OH DCA.Firstly,the weak expression of OleP was significantly improved using pRSFDuet-1 plasmid in the E.coli C41(DE3)strain.Next,the supply of heme was enhanced by the moderate overexpression of crucial genes in the heme biosynthetic pathway.In addition,a new biosensor was developed to select the appropriate redox partner.Furthermore,a cost-effective whole-cell catalytic system was constructed,resulting in the highest reported conversion rate of 6β-OH DCA(from 4.8%to 99.1%).The combinatorial catalytic strategies applied in this study provide an efficient method to synthesize high-value-added hydroxylated compounds by P450s.

Engineering caveolin-mediated endocytosis in Saccharomyces cerevisiae

As a potential substitute for fatty acids,common low-cost oils could be used to produce acetyl-CoA derivatives,which meet the needs of low-cost industrial production.However,oils are hydrophobic macromolecules and cannot be directly transported into cells.In this study,caveolin was expressed in Saccharomyces cerevisiae to absorb exogenous oils.The expression of caveolin fused with green fluorescent protein showed that caveolin mediated the formation of microvesicles in S.cerevisiae and the addition of 5,6-carboxyfluorescein showed that caveolae had the ability to transport exogenous substances into cells.The intracellular and extracellular triacylglycerol levels were then detected after the addition of soybean oil pre-stained with Nile Red,which proved that caveolae had the ability to absorb the exogenous oils.Lastly,caveolin for oils absorption and lipase from Bacillus pumilus for oil hydrolysis were co-expressed in the naringenin-producing Saccharomyces cerevisiae strain,resulting in naringenin production increasing from 222 mg/g DCW(dry cell weight)(231 mg/L)to 269 mg/g DCW(241 mg/L).These results suggested that the caveolin-mediated transporter independent oil transport system would provide a promising strategy for the transport of hydrophobic substrates.

Production of (2S)-sakuranetin from (2S)-naringenin in Escherichia coli by strengthening methylation process and cell resistance

(2S)-Sakuranetin is a 7-O-methylflavonoid that has anticancer,antiviral,and antimicrobial activities.Methylation process is involved in biosynthesizing(2S)-sakuranetin from(2S)-naringenin,in which S-adenosylmethionine(SAM)serves as the methyl donor.In this study,after methyl donor and substrate inhibition were identified as limiting factors for(2S)-sakuranetin biosynthesis,an efficient(2S)-sakuranetin-producing strain was constructed by enhancing methyl donor supply and cell tolerance to(2S)-naringenin.Firstly,PfOMT3 from Perilla frutescens was selected as the optimal flavonoid 7-O-methyltransferase(F7-OMT)for the conversion of(2S)-naringenin to(2S)-sakuranetin.Then,the methylation process was upregulated by regulating pyridoxal 5′-phosphate(PLP)content,key enzymes in methionine synthesis pathway,and the availability of ATP.Furthermore,genes that can enhance cell resistance to(2S)-naringenin were identified from molecular chaperones and sRNAs.Finally,by optimizing the fermentation process,681.44 mg/L of(2S)-sakuranetin was obtained in 250-mL shake flasks.The titer of(2S)-sakuranetin reached 2642.38 mg/L in a 5-L bioreactor,which is the highest titer ever reported.This work demonstrates the importance of cofactor PLP in methylation process,and provides insights to biosynthesize other O-methylated flavonoids efficiently in E.coli.