High-Yield Production Of FK228 And New Derivatives In Burkholderia Chassis

In recent years,cancer has become one of the three major killers that threaten human health.With the growth of Chinese population and the aggravation of aging,the number of cases and deaths of tumors ranks first in the world.Therefore,there is a huge demand for anti-tumor drugs in China’s clinical practice.Histone deacetylases(HDACs)are a class of proteases that play an important role in chromatin structure modification and gene regulation expression.Dysregulated expression of HDACs is one of the important causes of cell cancer.Histone deacetylase inhibitors(HDACi)have attracted attention as molecular targeted anti-tumor drugs that effectively inhibits the activity of HDACs,and have attracted people’s attention.FK228,also known as Romidepsin,is a natural product produced by Chromobacterium violaceum No.968 with histone deacetylase inhibitor(HDACi)activity.Its biosynthetic pathway has been preliminarily elucidated.FK228 belongs to a small family of uncommon cage-shaped bicyclic depsipeptides,which condenses two two-carbon structural units(malonylCoA)and five different types of amino acids by type I polyketide synthase(PKS)and nonribosomal peptidase(NRPS),respectively.As its structure contains a special disulfide bond,FK228 can specifically mediate a new anticancer mode of action:a reduced thiol "warhead"chelates the Zn2+in the catalytic pocket of HDACs,thereby inhibiting the activity of HDACs.FK228 has a significant effect on the inhibition of HDAC1 and HDAC2 in class Ⅰ HDACs.In 2009,FK228 was approved by the United States Food and Drug Administration(FDA)for the treatment of skin and peripheral T-cell lymphoma,and is currently the only known natural histone deacetylase inhibitor class antitumor drug.With the rapid development of bioinformatics and molecular biology technologies,other natural products in the FK228 family have been discovered,such as thailandepsins,chromopeptide A,and spiruchostatins.However,due to the low yield of industrial production strains of FK228,the chemical synthesis steps are tedious and its severe cardiotoxicity,there is an urgent need to develop an efficient synthetic biology platform to optimize the yield and structure of this family of drugs.Burkholderia thailandensis E264 is a gram-negative bacterium with fast growth rate and simple culture,is capable of expressing thailandepsin compounds in the FK228 family of compounds as well as a variety of other natural compounds with antibacterial and cytotoxic properties.Thus,the genome of the B.thailandensis E264 strain has been streamlined by deleting redundant genes and secondary metabolite biosynthetic gene clusters could reduce its metabolic background and avoid substrate competition between different gene clusters.Ultimately,it was modified into a chassis cell with high yields of FK228 family compounds.In this thesis,the tdp biosynthetic gene cluster(BGC)in the genome of B.thailandensis E264 was knocked out by a two-step homologous recombination strategy and the attB integration site was also introduced,resulting in a chassis cell called KOGC1.This strain lost the ability to produce thailandepsins.Based on this strategy,the malleilactone(mal)biosynthetic gene cluster was continued to be knocked out in the genome of the strain KOGC1,resulting in the construction of an ideal chassis strain named KOGC2(70.1 kb reduction in genome).The ability of this strain to produce FK228 was similar to that of KOGC1,but the metabolite background was less than that of chassis KOGC1.Heterologous expression of the "unnatural" biosynthetic gene cluster R-tdp-dep in the strain KOGC1,the final titer of FK228 can reach 581 mg/L,30-fold higher than the titer of the original strain C.violaceum No.968(19 mg/L),which is the highest known yield of FK228.Thus,this gene cluster was not only able to produce high yield of FK228,but also to express a new FK228 derivative,which was named FK228 B(1).The advantages of this chassis strain are:reducing the competition between the endogenous gene clusters and target gene clusters for background substrates,increase the copy number of target gene clusters,and increase the yield of target compounds.Secondly,the stability of the target gene cluster integration in the genome is enhanced,and also the unstable production of plasmid fermentation is avoided.Combinatorial biosynthesis is a strategy that involves the manipulation of relevant genes in microbial secondary metabolite biosynthetic pathways through operations such as substitution,knockout,and recombination,in order to produce "unnatural" natural products.This thesis mainly focused on the construction of a series of hybrid gene clusters based on three different biosynthetic gene clusters,such as thailandepsin(tdp)BGC,FK228(dep)BGC and spiruchostatin(spi)BGC,and heterologous expression in chassis cell KOGC2.We firstly replaced the A4-T4-E4-C5 domain units of the R-tdp-dep BGC with A4-T4-C5 domain units of the tdp BGC to obtain the hybrid BGC R-tdp-dep-M4(AT)M5(C)tdp via Redαβ recombineering plus CcdB counterselection method.In addition,R-tdp-dep-M4(ATE)M5(C)spi BGC and Rtdp-dep-M6(AT)tdp have also been constructed using the same method.Among them,the hybrid gene cluster R-tdp-dep-M4(ATE)M5(C)spi was able to produce six new FK228 derivatives,named FK228 C to FK228 H(2-7).The diversity of nonribosomal peptide structures is mainly determined by the specificity of the adenylation domain(A domain)in the NRPS module for recognizing different amino acid substrates.Therefore,the amino acid changes in modules 4 of compounds 2 and 3 are consistent with predictions.In module 4,the valine recognized by the acetylation domain of the FK228 gene cluster has changed to an alaine recognized by the acetylation domain of the spi gene cluster in compounds 2-5 and 7.However.the alaine in compound 2 and 3 have different configurations,possibly due to "jumping"translation caused by the epimerization domain(E domain)in module 4.Moreover,in compounds 4,5,6,and 7,different amino acids at position 7 simultaneously indicated that the A domain of module 7 of dep BGC is broadly selective for substrates.In addition,the other two hybrid gene clusters did not produce new compounds,which may be related to the linker sequence between the acetylation domain(A domain)and the condensation domain(C domain)between different gene clusters,and further verification and exploration are required.Furthermore,it is noteworthy that 2 and 3 were the main products from the strain KOGC2 harboring the BGC R-tdp-dep-M4(ATE)M5(C)spi and the titers of 2 and 3 reached up to 985mg/L and 435mg/L,respectively.The results of biological activity experiments on compounds 1-7 showed that compounds 2 and 3 had stronger cytotoxic activity than FK228.In addition,compound 3 exhibits stronger HDAC1 inhibitory activity as a prodrug than others.Therefore,mining new FK228 derivatives through NRPS module replacement provides a successful example for researchers to develop new FK228 family anticancer drugs.In polyketides,modification of the acyltransferase domain(AT domain)is also one of the important ways for mining new polyketides.Secondly,the ketoreductase domain(KR),dehydratase domain(DH)and enoyl reductase domain(ER)greatly enrich the structure of polyketides by controlling the degree reduction of β-carbonyl during the synthesis of polyketides.Among them,the KR domain performs NADPH-mediated reduction of β-keto groups to β-hydroxyl groups and determines the stereochemical structure of α-substituents andβ-hydroxyl groups.Currently,the mutational inactivation of the KR domain is also one of the strategies to study the function of KR and explore new polyketide compounds.In FK228 family compounds,the first two PKS modules mainly form the backbone and are generally not genetically manipulated.In addition,module 7 of PKS encoded by the tdpC2/spiC2 gene in the tdp and spi BGCs is different from FK228.The last extension unit of FK228 is valine,while a polyketide structure is existed in thailandepsin and spiruchostatin.By site-mutation inactivation of the KR domain in module 7 ofR-tdp BGC and relative hybrid BGCS,new FK228 derivatives have been discovered,increasing the structural diversity of FK228 family products and laying a solid foundation for future construction of "non-natural" natural product libraries of FK228.In this thesis,a new rational chassis cell was constructed using a Burkholderia-specific recombinant system,which could significantly improve the yield of FK228 and the efficiency of heterologous expression of target BGCs.The yield of FK228 reaches the highest known yield by this chassis cell,and the work laid the foundation for the industrial production of FK228.industrial production.New FK228 derivatives were mined through combinatorial biosynthesis in NRPS modules and site-mutation activation of the KR domain in PKS module.This work provides a feasible strategy for the development of novel histone deacetylase inhibitors as antitumor drugs.