Metabolic Engineering Of The Carbon Metabolism Network Focusing On Fatty Acid Metabolism To Enhance The Production Of Ten-membered Enediyne Tiancimycins

Enediyne natural products,characterized by the alkyne-alkenyl-alkyne structure,possess good antitumor activity and are the most active class of antitumor antibiotics discovered so far.The strong cytotoxicity of ten-membered enediyne tiancimycins（TNMs）makes it promising for the development of novel antitumor drugs,but the low yield limits further clinical applications.Streptomyces sp.CB03234-S is a tiancimycins（TNMs）high-producing strain obtained by streptomycin-induced ribosome engineering.The strain had improved the yield of TNMs to some extent,but it is still far from meeting the needs of industrial production.This paper was based on exploring the high-producing mechanism of TNMs in CB03234-S and used acetyl-Co A and malonyl-Co A,the precursors of TNMs biosynthesis,as key carbon metabolism nodes.We hope to further enhance TNMs production by enriching precursors through the regulation of carbon metabolic flow.The main research contents are as follows:By analyzing the transcriptomic data of the wild-type strain CB03234and the TNMs high-producing strain CB03234-S,it was found that the increase of carbon flux in CB03234-S was one of the reasons for the increased TNMs production.At same time,the fatty acid biosynthesis that also consumes acetyl-Co A was also significantly enhanced,while the corresponding fatty acid degradation metabolism was significantly weakened.Therefore,fatty acid metabolism is unfavorable for TNMs production as a precursor competition pathway.Since fatty acid biosynthesis is essential for the normal growth of bacteria,we started from fatty acid degradation metabolism of CB3234-S and reconstructed the pathway to redirect the diffluent acetyl-Co A to TNMs biosynthesis.We first divided five genes fad D、fad E、paa F、fad J and fad A encoding enzymes related to fatty acid degradation metabolism into two groups according to their expression levels.And fad E and fad A belong to a group with high expression（S-fad-2）,fad D,paa F,fad J belong to a group with low expression（S-fad-3）.These two sets of genes were overexpressed with promoter erm E and kas Op,respectively,while five genes were overexpressed together（S-fad-5）.The three mutants finally obtained effectively improved the TNMs production in different degrees.The mutant S-fad-3 increased the most and reached 21.0±1.2 mg/L,which was nearly 1.6-fold higher than that of CB03234-S.Further research found that the overexpression of fad D could reach same yield improvement effect as S-fad-3 and fad D encoding the first key enzyme in fatty acid degradation pathway.At the same time,we carried out the experiment of adding exogenous fatty acid during the fermentation process of recombinant strains.It was found that the biomass increased significantly while the TNMs production decreased,indicating that more acetyl-Co A produced by fatty acid degradation flowed to primary metabolism and interfered with secondary metabolism.Transcriptomic data also revealed differences in the expression of other enzymes related to precursors of TNMs biosynthesis in carbon metabolism.Phosphoenolpyruvate carboxykinase（PEPCK）is a key enzyme that promotes the re-entry of carbon flux from the tricarboxylic acid cycle into the glycolysis pathway,and the overexpression of its encoding gene pepck increased the yield of TNMs by 23%（16.3±0.4 mg/L）.The acetyl-Co A carboxylase（ACC）encoded by acc can convert acetyl-Co A to malonyl-Co A,and the overexpression of acc increased the yield of TNMs by 1.6-fold（20.7±1.1 mg/L）.Triacylglycerol（TAG）lipase is a key enzyme to initiate fatty acid degradation metabolism which can degrade intracellular TAG into single-chain fatty acids,and the overexpression of its encoding gene tag increased the yield of TNMs by 22%（16.2±1.9mg/L）.In addition,we also performed heterologous expression in CB03234-S.It was found that the overexpression of tag from Streptomyces albus ZD11 had a better yield improvement effect than the tag from CB03234-S.While the overexpression of acc from Streptomyces coelicolor M145 did not achieve the same effect as the acc from CB03234-S.Streptomyces albus ZD11 can utilize exogenous fatty acid efficiently,when the tag^ZD11derived from ZD11 was overexpressed in CB03234-S,the yield of TNMs increased by 1.5-fold（20.6±1.3 mg/L）.Previous study found that there is a possible copper-inducible bidirectional promoter in CB03234-S and Cu²⁺is closely related to the biosynthesis of TNMs.In order to avoid the adverse effects on bacterial primary metabolism caused by the persistent expression of above-mentioned key carbon metabolism enzymes induced by constitutive promoters.We expected to control the overexpression of these genes by the copper-inducible promoter derived from CB03234-S to synchronize them with TNMs biosynthesis.We verified the constructed copper-inducible promoter system based on different resistance genes,it was found that the system was not strictly regulated and there was background expression.No obvious difference was shown with or without the addition of Cu²⁺.On the other hand,due to the overexpression of fad D,acc³²³⁴,tag^ZD11 and pepck can improve the TNMs production when they were overexpressed individually.We also tried to overexpress these genes in combination to further improve the TNMs production.The results showed that expression of different combinations of these key primary carbon metabolism enzymes have different degrees of negative impact on bacteria growth.As a result,the yield of TNMs was lower than they were overexpressed individually.It also foreshadows the complexity of regulation on primary metabolism.Subsequent explorations need to focus on the selective and controllable expression of the above-mentioned genes and the reconstruction of key metabolic pathways.In this study,the acetyl-Co A precursor was enriched by regulating the carbon metabolism network focusing on fatty acid metabolism in CB03234-S to enhance the production of drug lead TNMs.And the reconstruction of the carbon metabolism pathway was explored preliminarily.Since acetyl-Co A is a general biosynthetic precursor of polyketide natural products,the regulation strategy on carbon metabolism in this study also provides a reference for the production improvement of other polyketide natural products.