Analysis of one-bond carbon-carbon coupling constants in [2-¹³C]acetate-derived penitrem A from Penicillium crustosum showed that [1,2-¹³C]acetate was formed during fermentation. P. crustosum was grown with [2-¹³C]acetate, and its 125.76 MHz proton noise decoupled ¹³C NMR spectrum revealed carbon-carbon couplings for several carbons, indicating eleven intact acetate units with an arrangement identical to [1,2-¹³C]acetate-derived penitrem A, along with interacetate and intermevalonate ¹³C-labelling. Indole carbon couplings were observed, possibly due to acetate conversion to glucose and entry into the shikimate pathway. The formation of [1,2-¹³C]acetate is attributed to [2-¹³C]acetate recycling in the Krebs cycle, a process unrecognized in low-field studies but detectable by high-field NMR. The carbon-carbon coupling information is valuable for biosynthetic and structural studies of fungal metabolites.