Biosynthesis Of Adipic Acid In Metabolically Engineered Saccharomyces Cerevisiae

Adipic acid is a very important chemical raw material.As a polymerization precursor of nylon 6,6,the market size of adipic acid continues to increase.The current chemical synthesis method has a long technological process,many by-products and serious emissions wastes from industry（N₂O）,relying on petroleum resources.The synthesis of adipic acid via biological method not only meets the chemical production concept of high efficiency and yield,but also meets the environmental protection requirements of low energy consumption and emission.In addition,the renewable resource can be used as alternative substrate for the synthesis of adipic acid,which is of great practical significance to get rid of dependence on petroleum resources.In this study,Saccharomyces cerevisiae was used as the starting strain to construct the reverse adipate degradation pathway（RADP）in the host by means of metabolic engineering,so as to realize the preliminary synthesis and product detection of adipate.The metabolic engineering and fermentation conditions were adopted to explore ways to improve the production of adipic acid.Also,the method of multiple copy site integration and screening harboring exogenous genes in S.cerevisiae was designed and attempted.Additionally,the way of high-efficiency synthesis of adipic acid by bio-chemical method was explored.The main results are as follows:（1）The de novo synthesis of adipic acid in S.cerevisiae was realized.The sequence of RADP gene from T.fusca was obtained and codon-optimized.Appropriate constitutive promoters and terminators were used to match Tfu＿0875,Tfu＿2399,Tfu＿0067,Tfu＿1647 and Tfu＿2576/7.The recombinant S.cerevisiae AA-1 containing reverse adipate degradation pathway was constructed by using the expression vector,and the preliminary qualitative detection of adipic acid was carried out by LC-MS.The LSC1-deficient strain was constructed with RADP yielding AA-3.However,the adipic acid production was only 0.33 mg/L.At the same time,obvious growth and metabolic retardation occurred.（2）The integration and screening method of multiple copies’integration for foreign genes in S.cerevisiae was designed and attempted.768 integrons were picked up from SD-URA plate to analyze the fluorescence intensity distribution.Integrons were selected in the range of high,medium and low fluorescence intensity for each 3 strains.The relationship between adipic acid titer and relative fluorescence intensity in high,medium and low copy integrons were analyzed.In conclusion,the strains with higher copy number（higher relative fluorescence intensity）obtained higher adipic acid production,and vice versa.（3）The genome integration strain was verified and fermentated in shake flask level.The genome integration strain AA-2 with RADP succeeded in an AA yield of 3.83 mg/L,which was12.9%higher than that of AA-1（3.39 mg/L）.In addition,the growth and metabolic rate of AA-2 strain were accelerated.Relative expression of pathway enzymes（AA-2 to AA-1）was determined via RT-PCR.The data shown that the expression of Tfu＿1647 increased by 96.79%in AA-2.The expression of Tfu＿2399,Tfu＿0067 and Tfu＿2576 increased,too.While the expression of Tfu＿0875 and Tfu＿2577 remained unchanged and decreased,respectively.（4）The levels of ATP and NADH in AA-2 cells were determined.Compared with wild-type strain,cellular ATP level of AA-2 strain increased significantly in the middle and later stage of fermentation.Different from the rapid decline of NADH level in wild-type strain,the proportion of cellular NADH of AA-2 increased slowly during the fermentation.（5）The effect of concentration of carbon source on the fermentation was investigated,and the 5-L level fermentation test was carried out.In terms of concentration of carbon source,a lower initial glucose concentration could reduce the Crabtree effect and production of ethanol,raising the yield of adipic acid.However,the titer will reduce.In 5-L level fermentation,a p H-controlled and constant-speed-feeding strategy was engaged,reaching an adipic acid yield of10.09 mg/L,which was the highest reported in the same host.（6）A new semi-synthetic bio-chem pathway to produce adipic acid was explored from glucose to adipic acid.S.cerevisiae strain Bga-4 containing VHb gene of bacterial hemoglobin was successfully constructed.The fed-batch fermentation showed that Bga-4 had higher sugar consumption efficiency,growth efficiency,by-product re-utilization rate and higher glucaric acid production.The 5-L level of glucaric acid fermentation was carried out in a process of high dissolved oxygen,free p H and constant glucose feeding rate.The titer of glucaric acid reached6.38 g/L,which was the highest reported.The chemical approach under mild condition from monosodium glucarate to adipic acid was realized.Ethyl glucarate-6,3-lactone was obtained by esterification and crystallization from potassium glucarate,which can be separated and extracted from fermentation broth.Then,ethyl glucarate-6,3-lactone was catalyzed by potassium perrhenate,palladium carbon and other catalysts under mild conditions.The conversion creates a novel and efficient synthetic route of adipic acid.