Study On The Synthesis Of Lycopene From Lactococcus Lactis By Metabolic Engineering

Lactic acid bacteria(LAB)as safe microorganisms have been used in food fermentation for a long time.They are also normal components of the intestinal flora,survive in the gastrointestinal tract and have multiple probiotic effects.Therefore,LAB are excellent chassis and delivery carriers for synthetic food additives,biotherapeutics and other functional products.Lycopene is a terpenoid compound composed of 8 isoprene units.As one of the most strongest antioxidant,lycopene can play a variety of physiological functions such as antioxidant,radiation resistance,cancer prevention and so on when it get into the body.The synthesis of lycopene using food-grade LAB as hosts have shown a great potential for application due to the advantages of safety and its can be taken in the form of living bacteria without extraction.Lactococcus lactis is the model strain of LAB,which with clear genetic background,relatively simple metabolic pathway,and mature operating tools.Its application scope has been extended from the field of food fermentation to the microbial "cell factory" that produce high value-added products such as polyols and diacetyl.Due to the complex metabolic pathway of lycopene,there have been few reports on the synthesis of lycopene in Lc.lactis,and a large number of lycopene synthesis in Lc.lactis has not been realized at present.In this paper,Lc.lactis NZ9000::loxp was used as the starting strain to optimize the expression of crtEBI gene from Pantoea ananatis and as stable recombinant strain to synthesize lycopene.The yields of lycopene per unit cell was increased by controlling fermentation conditions and modifyingmetabolic pathways.We also verified that lycopene synthesized by Lc.lactis had the physiological functions of anti-oxidation.The specific experimental results are as follows:1.Construction of recombinant Lc.lactis for synthesizing lycopene.The synthesis of lycopene in organism needs to go through multi-step reaction.The first step is the synthesis of famil pyrophosphate from mevaluanoic acid through multi-step enzyme catalysis.Farnesyl pyrophosphate is further catalyzed by the rate-limiting enzyme geranyl geranyl pyrophosphate synthase CrtE,phytoene synthase CrtB,and phytoene dehydrogenase CrtI to produce lycopene.It is predicted that the lycopene synthesis pathway encoded by the Lc.lactis NZ9000 is incomplete and lacks crtB and crtI genes.To realize the production of lycopene in Lc.lactis,we used the shuttle vector pLEISS of Escherichia coli and LAB and realized the inducible and constitutive expression of crtEBI gene in strains Lc.lactis NZ-crtEBI and Lc.lactis NZ-P207-crtEBI,respectively.The Crelloxp site-specific recombination system was used to realized inducible and constitutive expression of crtEBI gene in strains Lc.lactis NZ-int-crtEBI and Lc.lactis NZ-int-2Pldh-crtEBI,respectively,with the crtEBI gene integrated on the chromosome.After the recombinant strains NZ-crtEBI and NZ-int-crtEBI were successively subcultured for 100 generations under conditions of no selective pressure,the recombinant plasmid carrying the crtEBI gene in the strain NZ-crtEBI and 15 copies of crtEBI gene on the genome of strain NZ-int-crtEBI both remained stable,which proved the genetic stability of the two recombinant strains.The results of high performance liquid chromatography showed that the yields of lycopene in the NZ-crtEBI and NZ-P207-crtEBI achieved 0.59±0.00 and 0.11±0.00 mg/L,respectively;the yields of lycopene in the NZ-int-crtEBI was 0.54±0.04 mg/L,and the NZ-int-2Pldh-crtEBI did not synthesize lycopene.The molecular weight and size of the extracted and expressed lycopene were consistent with the lycopene standard by mass spectrometry analysis,indicating that we successfully realized the expression of lycopene in Lc.lactis.The relationship between the lycopene production and biomass of 9 randomly selected recombinant NZ-crtEBI was analyzed.We found that the higher the yields of lycopene,the lower the biomass of the bacteria,this indicates that there is a competitive relationship between the growth of Lc.lactis and the synthesis of lycopene.2.Optimization the fermentation conditions for the synthesis of lycopene in Lc.lactis.Lactococcus lactis is a facultative anaerobic microorganism,but in the presence of heme can carry out aerobic respiration,which will improve the density of bacteria.In order to increase the bacterial biomass.Firstly,the fermentation medium of NZ-crtEBI was optimized by orthogonal experiment.The results showed that the lycopene yields was 0.83±0.10 mg/L and the cell density was 1.90±0.05 under the conditions of 1%glucose,0.5%yeast powder and 0.5%peptone,the yields was increased by 40.6%compared with that before optimization.The contents of lactic acid,acetic acid,diacetyl and acetoin were 84.05±0.22 mM,24.33± 1.26 mM,4.06±0.00 mM and 1.25±0.19 mM,respectively.On this basis,4 pg/mL heme was added into the medium,and the conditions such as initial pH,fermentation temperature and shaker speed were further optimized.The results showed that the lycopene yields of NZ-crtEBI reached 1.09±0.01 mg/L fermented at pH 6.5,140 rpm,30? for 12 hours,and increased by 31.33%;the OD600 of NZ-crtEBI reached 3.17±0.01,and increased by 66.84%.In addition,the contents of lactic acid,acetic acid,diacetyl and acetoin were 61.80±2.58 mM,26.13±0.05 mM,4.21±0.14 mM and 11.39±0.31 mM,respectively,indicating that the cell density and lycopene yields were significantly increased under aerobic condition(P<0.05).The scale-up experiment was carried out in a 5 L fermenter.The recombinant strain NZ-crtEBI was fed with 30%glucose as carbon source for fermentation.After fermentation at pH 6.5 and aeration rate of 1 L/min for 24 hours,the lycopene production reached 2.01±0.46 mg/L and the OD600 increased to 6.12±0.11.3.Blocking the competitive pathway of lycopene precursor acetyl-CoA.Acetyl-CoA is the synthetic precursor of lycopene.Acetyl-CoA is produced by pyruvate under the catalysis of pyruvate dehydrogenase system in Lc.lactis.At the same time,pyruvate also produces lactic acid and ?-acetolactate catalyzed by lactate dehydrogenase(LDH)and a-acetolactate synthase(ALS),respectively.Therefore,blocking the LDH and ALS pathways will accumulate more acetyl-CoA in cells,which is conducive to improving lycopene production.Using CRISPR-Cas9-assisted ssDNA recombineering technology to knock out the lactate dehydrogenase gene llmg₁120 in the genome of Lc.lactis NZ9000::loxp,or deleted the ?-acetolactate synthase gene llmg₁309 at the same time,to obtain the knockout strains Lc.lactis NZ?ldh and Lc.lactis NZ?ldh?als,respectively.The recombinant strains NZ?ldh-crtEBI and NZ?ldh?als-crtEBI were constructed by electrotransforming the inducible recombinant plasmid pLEISS-crtEBI.The yields of lycopene per unit of recombinant strains NZ-crtEBI,NZ?ldh-crtEBI and NZ?ldh?als-crtEBI were 0.37,0.42 and 0.43 mg/L/OD600,respectively,without heme;and 0.31,0.5 and 0.4 mg/L/OD600,respectively,with heme.Combined with the quantitative findings of metabolites such as lactic acid,acetic acid and acetoin,we found that blocking the LDH pathway forced part of the carbon to flow to lycopene and acetoin,while blocking the LDH and ALS pathways allowed more carbon to flow to lycopene.The results showed that blocking the LDH and ALS pathways contributes to the accumulation of lycopene in cells.In addition,when compared with NZ-crtEBI,the recombinant bacteria NZ?1dh-crtEBI and NZ?ldh?als-crtEBI with or without heme,the biomass is significantly reduced,and the NADH/NAD+level is significantly increased,the indicated that the intracellular redox imbalance affects cell growth after blocking the LDH pathway.4.Antioxidant effects of lycopene synthesized by Lc.lactis.Lycopene is a natural lipid soluble pigment and has a good antioxidant activity.In this chapter,lycopene concentrate synthesized by Lc.lactis NZ-crtEBI was prepared by rotary evaporation,and its in vitro scavenging rates for DPPH,O2+ and OH were determined.When lycopene concentrations were 5 g/mL,the scavenging rate of DPPH and O2+ was more than 50%and 80%respectively.When the concentrations were 10 ?g/mL,the scavenging rate of ·OH was more than 20%,and the antioxidant capacity was significantly higher than that of Vc,VE and BHT at the same concentrations.Furthermore,the ability of lycopene synthetic strain NZ-crtEBI to resist H2O2 stress was further explored.The results showed that the survival rate of NZ-crtEBI was significantly increased compared with the control strain NZ-pLEISS after 2 h of 2 mM,4 mM and 6 mM H2O2 stress,which proved that NZ-crtEBI has the ability to resist oxygen stress.To explore the protective effect of lycopene recombinant bacteria on oxidative stress of intestinal epithelial cells.The wild strain NZ9000::loxp,the control strain NZ-pLEISS and the the lycopene synthetic strain NZ-crtEBI were co-cultured with intestinal epithelial cells NCM 460,respectively.After co-cultured with and NZ9000::loxp and NZ-pLEISS,the survival rate of NCM 460 after 4 h under H2O2 stress both was 29.55%±2.56 and,30.50%±2.35,respectively,while after co-cultured with NZ-crtEBI,the survival rate after 4 h was 46.5%±5.20,which was significantly improved(P<0.01).These results indicated that lycopene could effectively protect cell NCM 460 from ROS damage,and fluorescence positive microscope observation results confirmed that lycopene synthetic strain reduced ROS level in cell NCM 460.