Chlorothricin (CHL), produced by Streptomyces antibioticus DSM 40725 (wild-type strain, WT), belongs to a growing family of spirotetronate antibiotics that have biological activities inhibiting pyruvate carboxylase and malate dehydrogenase. ChlF2, a cluster-situated SARP regulator, can activate the transcription of chlJ , chlC3 , chlC6 , chlE1 , chlM , and chlL to control CHL biosynthesis. Co-expression of chlF2 and chlK encoding type II thioesterase in WT strain under the control of P kan led to high production of chlorothricin by 840% in comparison with that of WT. Since the inhibitory activity of CHL against several Gram-positive bacteria is higher than des-CHL, combinatorial strategies were applied to promote the conversion of des-CHL to CHL. Over-expression of chlB4 , encoding a halogenase, combining with the supplementation of sodium chloride led to further 41% increase of CHL production compared to that of F2OE, a chlF2 over-expression strain. These findings provide new insights into the fine-tuned regulation of spirotetronate family of antibiotics and the construction of high-yield engineered strains.