Production Of 5-aminolevulinic Acid By Escherichia Coli Using C4 Pathway

As the precursor of almost all kinds of tetrapyrrole compounds,i.e. heme, vitamin B12, chlorophyll,5-aminolevulinic acid has broad application prospect in agricultural and medical field. In the area of agriculture, ALA could promote the photosynthesis and tissue differentiation, kill dicotyledon weeds selectively without harming the crops and enhance the cold resistance and salt resistance of plants. In the field of medicine, ALA has produced a remarkable effect in diagnosis and treatment of cancer as a kind of photo dynamic therapy medicine; ALA has been greatly recognized in treating mycosis and rheum arthritis; ALA also has a certain effect in beauty and skincare. In general, ALA has great value of engineering application and industrial prospect.The traditional chemical synthesis method for ALA has the following disadvantages:high cost, low yield, high price and severe environmental pollution. The biological methods has the advantages of simple production process, great production potential and environmental safety. Two main biosynthetic pathways have been used for ALA:one is C5 pathway, ALA was synthesized with glucose as the sole carbon source, which was first developed by our group, but this pathway involved more enzymes, and the regulation was complicated, thus making the increase in yield hard to accomplish; the other one is C4 pathway, ALA synthetase catalyzed succinyl-CoA and glycine to form ALA in photosynthetic bacteria and Escherichia coli, as the pathway was short and the control of it was simple, it was widely studied. However, the addition of expensive precursors such as succinyl-CoA and glycine, aeration and agitation for aerobic fermentation all lead to high production cost. It is clear that the key point is to optimize the C4 pathway to improve the production and yield as well as lower the production cost.Firstly, this research focused on the optimization of the C4 pathway in aerobic condition. The methods include:selecting strains with different genotypes backgroud for aerobic fermentation, comparing their production capacities and analyzing the related metabolism; increasing the dissolved oxygen; optimization of ALA synthetase from different sources. And we found that, different strains showed different ALA synthesis capacity and growth state. E.coli KNSP was able to utilize fatty acid and glucose as carbon sources. When ALA synthetase from Rhodopseudanonas palustris KUGB306 was expressed in KNSP, the resulting strain KRPA could produce 3.61g/l ALA in shake flask and also exhibited good growth and high vigor. This part clearly demonstrated that ALA production capacity is dependent on the strain backgroud. Strains with different genetypes show diverse metabolic flux, inconsistent ALA synthetase enzyme activities and different trends of transformation to downstream products, which will surely lead to differentiated ALA yields.Secondly, for the first time this research established microaerobic and aerobic-anaerobic fermentation system for ALA production. The previously built strain YL106 could produce abundant succinate in a whole-phase fermentation system,i.e.aerobic, microaerobic and anaerobic, and the by-products such as lactate and acetate were low. When ALA synthetase from Rhodobacter sphaeroides was ovexpressed in YL106, the resulting strain YL106A could produce 5.88 g/1 ALA in shake flask and the yield was 0.557 g ALA/g glucose. YL106A produced 7.67 g/1 ALA in the 5L fed-batch fermentation experiment. To verify whether the transformation of succinate to succinyl-CoA was a limiting step, catl or sucCD were over expressed in YL106A. We found that the overexpression of cat] could significantly improve the ALA yield and production rate in microaerobic condition. These strains will hopefully lower the aeration and agitation cost and simplify the downstream separation and extraction process.Finally, to reduce the cost of ALA biosynthesis, the precursors succinyl-CoA and glycine should be synthesized with glucose as the sole carbon source, so we could get ALA directly from glucose without further addition of expensive precursors. The previously constructed serine-producing strain 3Sp could accumulate 4.5g/l serine. Serine could be rapidly transformed into glycine, and the latter is one of the precursor of ALA. The sucD gene was knocked out in serine-producing strain 3Sp, so succinate addition became unnecessary. Then ALA synthetase was overexpressed, ALA was produced. Different ALA synthetases were also screened, various cofactors such as folate, PLP and glutamtate were optimized and the final strain 5SRPO could accumulate 208mg/l ALA. For the first time the strategy of producing ALA directly from glucose without relying on succinate and glycine was built and will remarkbly lower the cost of ALA production.These results will shed a new light on acquisition of new strains and improvement of ALA production and yield as well as reduction of cost.