Applied And Basic Research On Microbial Conversion Of Glycerol Into 1,3-propanediol And Lactate

Driven by energy insecurity and environmental concerns,sustainable production of chemicals,fuels,and materials has drawn increasing attention recently.To provide these sustainable solutions,utilizing renewable bioresources to produce fuels and chemicals by microbial cell factories are required,accompanied with the rise of bio-refinery and bio-manufacturing.However,the raw materials used by bio-refinery and bio-manufacturing are mainly sugars,such as glucose.In this case,the utilization of sugar not only competes with people for food,but also increases the cost of bio-fuel and bio-chemical production.Therefore,it is important to replace sugar substrates with non-food substrates.In recent years,with the development of biodiesel industry,production of glycerol increases dramatically and its price decreases gradually,which makes glycerol an ideal feedstock for bio-production.Moreover,the degree of reduction per carbon of glycerol is higher than carbohydrate-based feedstocks such as glucose,suggesting that glycerol could provide more reducing power for products formation.Therefore,conversion of glycerol to fuels and chemicals by microbes has attracted more and more attention.There are also some successful commercial cases.The platform chemical 1,3-propanediol can be used as solvents,lubricants,antifreeze,adhesives and protective agent,and it has extensive applications in polymers,food,medical and cosmetics production.Of particular interest is its use for producing polytrimethylene terephthalate(PTT),which has superior stretching and stretch recovery characteristics.Lactate was selected as one of the promising platform chemicals by the U.S.Department of Energy,and is an important monomer for producing poly-lactate(PLA).PLA is regarded as the substitute for petroleum-derived plastics as its biodegradable,biocompatible and environmentally friendly characteristics.Because there are limitations in the chemical synthesis of 1,3-PD and lactate,bio-production of these two chemicals has drawn increasing attention.The pathways,bacteria and fermentation process of 1,3-PD and lactate production have been always the hot research topics.However,there are still some problems such as the low concentration of products and the low utilization efficiency of substrate in current bio-production technologies and methods.The conducting basic and applied basic research in microbial conversion of glycerol to 1,3-PD and lactate will be helpful for the understanding of glycerol metabolism and eliminating the restrictive factors in 1,3-PD and lactate production,thereby realize the high-level production of these two chemicals by building more effective cell factories using metabolic engineering.In the present study,we performed the genome sequencing of production strains,constructed engineered strains for co-production of 1,3-PD and lactate,and carried out co-fermentation to solve the existing problems in 1,3-PD and lactate bio-production.More details are as follows:Firstly,the genomes of 1,3-PD producing strains screened by our lab,including Klebsiella oxytoca PDL-0 and Clostridium baratii XCM,and the standard strains including C.butyricum 10702 were sequenced.The draft genome assembly reached 6.55 Mb,3.08 Mb and 4.59 Mb,respectively.Besides,the Whole Genome Shotgun project of C.butyricum 10702 has been deposited at DDBJ/EMBL/GenBank under the accession number AQQF00000000.In addition,all the genome annotation of these three bacteria has been finished by the RAST server.And the glycerol metabolism genes and 1,3-PD producing genes in dha operon were analyzed.Based on the data from NCBI,we aligned the dha operons of Klebiella oxytoca PDL-0,Clostridium baratii XCM,and C.butyricum 10702.The result showed that dha operon could be classified into 3 types.First,1,3-PD producers belong to Klebsiella genera and Citrobacter genera.Second,some 1,3-PD producers belong to Clostridium genera,such as C.baratii XCM and C.pasteurianum DSM 525.Third,other 1,3-PD producers belong to Clostridium genera,such as C.diolis DSM 15410 and C.butyricum DSM 10702.Moreover,according to the result of the evolutionary tree of dha operon,although both of the second and the third type belong to Clostridium genera,the amino acid sequences of the second type have higher identity to the first one,and the ways genes arranged are very similar.All these indicate that the evolutionary relationship of the second type is closer to the first one,which may be due to the different origins for acquiring dha operon and 1,3-PD production ability in different microbes.Secondly,to improve the efficiency of glycerol utilization and decrease the production of by-products,the strategy of co-production of 1,3-PD and optical pure lactate was presented on the premise of developing a rebalance between glycerol reduction and oxidation branches.To select a favorable oxidized chemical for co-production with 1,3-PD,a series of criteria should be fulfilled.First,a chemical with high commercial value and important applications is preferred.Second,the biosynthetic pathway of the oxidized chemical should minimize loss of carbon to achieve maximum carbon conservation.Third,co-production of the oxidized chemical with 1,3-PD should help maintain the intracellular redox balance,which is crucial for cell survival and metabolism.Fourth,a chemical with good biological compatibility is favorable,as this affords high-yield and long-term bio-production.Fifth,the co-product should be separable from the fermentation broth and 1,3-PD easily in order to render the downstream processing economical and efficient.After deliberating on these criteria,the lactate-producing pathway was chosen for coupling with the 1,3-propanediol-producing pathway.Then,the 2,3-butanediol,ethanol,acetate,and succinate pathways were blocked in sequence,leading to a series of metabolic engineered strains,of which PDL-5(K.oxytoca PDL-0 ?budA?budB?adhE?ackA-pta?pox?frdA)highly co-produced PD(76.2 g/L,2.5 g/L·h)and D-lactate(111.9 g/L,3.7 g/L·h)through bioprocess engineering.The total conversion yield of PD and D-lactate reached 0.95 mol/mol glycerol.Thirdly,to realize the co-production of 1,3-PD and optical pure L-lactate,K.oxytoca strain PDL-5 was engineered.First,the d-LDH encoding gene IdhD of strain PDL-5 was replaced with the l-LDH encoding gene ldhL from Lactobacillus casei ATCC 334(ldhLLc),resulting in strain PDL-7,which only produced small amounts of L-lactate.Besides,pyruvate accumulation could be observed,which may be attributable to the fact that the l-LDH activity of this strain was not high enough.To increase the enzyme activity,l-LDH encoding genes from L.casei ATCC 334,K.oxytoca PDL-0,and B.coagulans 2-6 were cloned and induced to express under the control of Ptac,of which the ldhLB,gene showed the best performance.Finally,to bypass the use of IPTG,ldhLBc was then inserted into the chromosome of strain PDL-7 at the budAB locus and under the control of the constitutive promoter Pbud.The resulting strain PLL co-produced 70.0 g/L of PD and 104.0 g/L of L-lactate,with the conversion yield 0.42 mol/mol and 0.53 mol/mol,respectively.The total conversion from glycerol achieved 0.95 mol/mol.Compared to aerobic or microaerobic fermentation,Clostridium sp.use the coenzyme B 12-independent 1,3-PD biosynthetic pathway and can convert glycerol into 1,3-PD anaerobically.At the same time,by-products such as acetate,butyrate and butanol are also produced with 1,3-PD.However,no genetic tools have been available for C.butyricum DSM 10702 and C.diolis DSM 15410,making the metabolic engineering of these strains difficult.The low conversion yield of 1,3-PD and lots of by-products formation have been the main factors limiting anaerobic fermentation.In this section,for effective and low-cost 1,3-PD production,the potential use of lignocellulosic hydrolysates as co-substrates by C.diolis DSM 15410 was investigated.First,as lignocellulosic hydrolysates is mostly glucose,xylose,and arabinose,therefore,these three individual sugars were used,separately,as co-substrates with glycerol,in 1,3-PD production by a Clostridium diolis strain DSM 15410,resulting in an 18%-28%increase in the 1,3-PD yield.Then,co-fermentation of the mixed sugars and glycerol increased the 1,3-PD yield by 22%relative to fermentation of glycerol alone.Moreover,a higher intracellular NADH/NAD+ ratio was achieved,and the transcription level of dhaD decreased,which were benefit for 1,3-PD production.Thereafter,two kinds of lignocellulosic hydrolysates,corn stover hydrolysate and corncob molasses,were individually co-fermented with glycerol.The maximum 1,3-PD yield from glycerol reached 0.85 mol/mol.Fed-batch co-fermentation was also performed,improving the 1,3-PD yield(from 0.62 mol/mol to 0.82 mol/mol).Fed-batch fermentation of crude glycerol and corn stover hydrolysate can also produce 1,3-PD at the same level.All these results demonstrate that utilization of lignocellulosic hydrolysates as co-substrates is-an efficient and economical way to biosynthesize 1,3-PD.Finally,the sequence analysis and protein crystallization of LldR of Pseudomonas aeruginosa XMG,which is an FadR-type regulator,were investigated.First,sequence network analysis of the LldR proteins from different bacterial species,including Escherichia coli and P.aeruginosa were performed.The result showed that LldR proteins from Pseudomonas sp.and Escherichia coli were separated into different clusters,suggesting that LldRs are derived from two ancestors that functionally diverged.Then,the sequences of amino acids of three LldR homologues,including LldR from P.aeruginosa XMG,E.coli and C.glutamicum,were aligned,and the amino acid residues indispensable for DNA-binding and Zn2+-binding were indicated.Third,the prediction of the secondary and third structures of PLldR from P.aeruginosa and ELldR from E.coli were performed and compared with CLldR from C.glutamicum.The results indicated that the N-terminal domain of PLldR,ELldR and CLldR are more conversed than C-terminal domain.Finally,the recombinant PLldR protein(LldR of P.aeruginosa)was expressed,purified,and crystallized.Preliminary X-ray diffraction analysis of LldR protein crystals was performed.The PLldR crystal diffracted to 2.55 A resolution and belonged to the trigonal space group P3,with unit-cell parameters a = 68.5 A,b = 68.5 A,and c = 237.0 A.These results will facilitate further understanding of the regulatory mechanism and the adaptation to sensing of both L-lactate and D-lactate of LldR proteins from Pseudomonas sp.in lactate metabolism.