| 5-Hydroxymethylfurfural?HMF?is a high-value bio-based platform compound that can be prepared via dehydration of carbohydrates.HMF contains active functional groups such as furan ring,primary hydroxy and formyl groups,which can be oxidized and reduced into a series of valuable chemical intermediates.2,5-Bis?hydroxymethyl?furan?BHMF?is a reduction product of HMF,and is also an important building block in the synthesis of drugs,crown ethers,functional materials,and bio-based polymers,etc.Currently,chemical methods remains dominant in the synthesis of BHMF;nonetheless,they are characterized by harsh reaction conditions,high energy consumption,unsatisfactory selectivities,and environmental infriendliness.On contrary,the biocatalytic processes are performed under mild conditions,and are efficient,selective and environmentally friendly.Therefore,they may be a good option to replace the traditional methods for the synthesis of BHMF.Previously,a strain Meyerozyma guilliermondii SC1103 which can tolerate a moderate concentration of HMF and selectively synthesize BHMF was isolated from soil samples by us,but unsatisfied catalytic efficiency and substrate tolerant level remained.It is expected that recombinant strains overexpressing key enzymes may exhibit the improved catalytic performances.In this thesis,therefore,the key enzymes that are responsible for the HMF reduction were mined from M.guilliermondii SC1103 genome,cloned,expressed and biochemically characterized.Then,these enzymes were introduced into Saccharomyces cerevisiae,and an engineered S.cerevisiae capable of tolerating high concentrations of HMF was successfully constructed.An efficient and selective biocatalytic approach was established for the synthesis of BHMF.Five putative alcohol dehydrogenase?ADH?genes including adh434,aad938,aad1669,adh3195 and adh3695 were present in Meyerozyma guilliermondii SC1103 cells.These genes were cloned into the expression vector pET-28a?+?,followed by transformation into Escherichia coli BL21.Unfortunately,these enzymes were expressed in the form of inclusion bodies.In addition,no improved expression of soluble proteins was achieved by using various methods.Then,these ADH genes were cloned into the expression vector pPICZ?A,followed by electro-transformation into the genome of Pichia pastoris X-33 strain.The results showed that all the proteins could be solubley expressed.Upon purification,their properties were studied.It was found that all ADHs preferred NADH as their coenzymes.Their optimum reaction temperature was more than 45°C;among ADHs tested,ADH3195 displayed the highest stability,and this enzyme retained 75%of residual activity after incubation for 5 h at40°C.The optimal pH values of ADHs were 5-6,except for AAD938 with optimal pH being 7.Of ADHs examined,AAD1669 exhibited the highest activities?0.24-0.29 U/mg?when HMF and furfural worked as substrates.Most metal ions had a slight effect on the activity of AAD1669.The five ADH genes were cloned into the expression vector pYES-2,and then transformed into S.cerevisiae INVSc1.Five engineered yeast strains were successfully constructed for the BHMF synthesis.Of recombinant strains,recombinant S.cerevisiae INVSc1-aad1669 showed the high catalytic performances in the reduction of HMF.This recombinant strain showed good activities under pH 5-9 and 30-40°C.Compared to the wild-type yeast,this recombinant strain exhibited higher activities as well as stronger tolerance towards substrate and product.The cell viability was more than 75%after the recombinant strain was incubated in the presence of 250 mM substrate for 12 h.W hen the BHMF concentration was up to 400 mM,no significant product toxicity toward the cells was observed,and the product inhibitory effect on the biocatalyst was weak.Under the optimal reaction conditions,HMF of 200-250 mM could be reduced into BHMF within 9-24 h by this recombinant yeast,with the yields of 91-94%and the selectivities of 99%.In addition,BHMF of 345 mM was produed within 22.5 h by using a fed-batch strategy,providing a space-time yield of approximately 46 g·L-1·d-1.It was feasible to replace glucose with inexpensive corncob hydrolysates as co-substrate for the HMF reduction by this strain,and the BHMF yields and selectivities were comparable with the above two co-substrates.In this study,the key enzyme genes responsible for the HMF reduction were successfully mined from M.guilliermondii SC1103 genome,and their enzyme properties were studied,which might lay the foundation for their applications in synthetic chemistry.The features and laws in selective reduction of HMF by S.cerevisiae INVSc1-aad1669 were preliminarily elucidated,which might enrich the theoretical knowledge for biocatalytic valorization of bio-based platform chemicals. |
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