Breeding Of High-yield L-isoleucine Strains And Research On Fermentation Characteristics

As one of the eight essential amino acids in the human body,L-isoleucine plays an important role in human life metabolism due to its special structure and function,so it is widely used in the fields of medicine,cosmetics,food and feed.There are three production methods of L-isoleucine,namely protein hydrolysis,chemical synthesis and fermentation.At present,the most common industrial application is fermentation.The selection of high-yield L-isoleucine strains and the optimization of the fermentation control mode are the key to the production of L-isoleucine by fermentation.Based on the theoretical basis of the biosynthetic pathway of L-isoleucine,a reasonable design of screening methods and optimized fermentation mode can quickly obtain high-yield L-isoleucine strains and increase the production of L-isoleucine.The acquisition of high-yield L-isoleucine strains and fermentation regulation based on metabolic pathway analysis are of great significance to the realization of L-isoleucine industrialization.This article has conducted the following four aspects of research:(1)Establish a high-throughput screening strategy for sulfaguanidine resistance marker and ninhydrin multiwell plate detection.After 180 s of ARTP treatment,Corynebacterium glutamicum 23798 was inoculated into the culture medium containing 0.4 mg/m L sulfanidine for 2-3 days.The strains with large single colonies were selected and then inoculated into the liquid culture medium with 24 well plates.A dominant mutant strain,Corynebacterium glutamicum B1,was bred by using the specific reaction of amino acid with ninhydrin and the corresponding fermentation supernatant of the strain by 96-well high-throughput screening.The L-isoleucine yield of the strain was 62.0% higher than that of the original strain when cultured in 500 m L shaking flask for 48 h,and its genetic traits were stable.However,the screening method established in this chapter has a long period and is labor-intensive,so it is not suitable for large-scale screening.The screening method will be improved in the later stage.(2)Establish a new method for screening high-yield L-isoleucine mutant strains.The newly invented droplet generator combined with the screening method of adding a critical lethal concentration of α-aminobutyric acid to the microplate,and successfully screened a dominant mutant strain Corynebacterium glutamicum K5 with a 16.2% increase in L-isoleucine production.Compared with the method of selecting strains in Chapter 2,the new screening method established in this chapter has shortened the screening cycle by nearly half,and the efficiency has been significantly improved.However,Corynebacterium glutamicum K5 was cultured in fermenter,and the yield of L-threonine reached 3.5 g/L,which was relatively high.By analyzing the biosynthetic pathway of L-isoleucine,L-threonine dehydrase could convert L-threonine into α-keto-butyric acid,then L-isoleucine was produced.Indicating that the activity of threonine dehydrase needs to be strengthened.(3)The effect of adding different concentrations of the precursor substancesα-aminobutyric acid and α-ketobutyric acid on the fermentation properties of L-isoleucine was investigated in different periods.According to the experiment,whenα-aminobutyric acid and α-ketobutyric acid were added at 8 h of culture,the molar conversion rate of both reached the highest,reaching 42.5% and 84.1% respectively,which may be the growth state of the bacteria at this time.Best,the vitality of related enzymes in the body is also the most vigorous.At the same time,the maximum molar conversion rate achieved when α-ketobutyric acid is added is nearly double the maximum molar conversion rate achieved when α-aminobutyric acid is added.This shows the conversion effect of α-ketobutyric acid Better,it can also be judged that the deamination ability of Corynebacterium glutamicum K5 needs to be improved.With the increase of the concentration of α-aminobutyric acid and α-ketobutyric acid,the yield of L-isoleucine is increased,but the molar conversion rate is significantly reduced,indicating that simply adding precursor substances is not essential To improve the ability of the strain to produce L-isoleucine,it is necessary to start from the strain’s own related enzymes to increase the ability of its own metabolism to produce α-ketobutyric acid,thereby enhancing the ability to produce L-isoleucine.(4)Study on the relationship between threonine dehydratase and L-isoleucine synthesis.By using the Corynebacterium glutamicum K5 genome as a template and designing primers,after amplifying the control gene of threonine dehydratase,it was ligated with the plasmid PET-28 a to successfully construct a recombinant plasmid with the control gene of threonine dehydratase.The recombinant plasmid was successfully expressed after being transformed into E.coli,and its enzymatic activity reached 2 U/m L.Threonine dehydratase can catalyze the formation of α-ketobutyric acid from threonine to produce L-isoleucine.If the total activity of threonine dehydratase in the bacteria is increased,it will be beneficial to the production of L-isoleucine.If the recombinant plasmid is introduced into Corynebacterium glutamicum K5 by electroporation to induce its expression,it may further increase the production of L-isoleucine.