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The filamentous fungus Trichoderma reesei is a well-known source of cellulase enzymes in many industrial applications. Nevertheless, its use as a host for heterologous expression is limited. In this study, many promoters were tested in T. reesei by expressing eGFP, including PamyB, Ppki, Ppdc, PgpdA, and Peno, by an established set of vectors. A remarkable fluorescence level was achieved in the case of Ppdc, PgpdA, and Peno, a moderate level with Ppki, and a weak emission for PamyB. In addition, 6-hydroxymellein (6-HM) 82 was expressed exclusively in T. reesei under the control of the native Ppki. as an example of fungal secondary metabolites. Many dimerized sorbicillinoids have been identified in this study to be produced by T. reesei, some of which were fully confirmed by 2 D NMR including, spirosorbicillinol B 123. The sorbicillin BGC in T. reesei was ascertained during this study by knockout NR-PKS (sorA). Furthermore, scytolide 128, a natural herbicide, was confirmed to be produced by T. reesei for the first time. Targeted knockout of two genes encoding a short-chain dehydrogenase-reductase (SDR) and a cytochrome P450 monooxygenase confirmed they have no function in the dimerization reactions. Targeted knockout of the Flavin-dependent monooxygenase (FMOII, SorD) has diminished dimers’ production. Two engineered strains were established, T. reesei Ppki·hfas, and T. reesei Ppdc·hfas, in which the Homo sapiens FAS (hFAS) is expressed under the control of the native constitutive promoter of pki and pdc, respectively. The strain Ppdc·hfas can produce the highest FAMEs reaching 544 mg/kg directly from rice straw, presenting the ability of T. reesei as an environment-friendly biocatalyst in converting agricultural- biomass into biodiesel substrates (FAMEs such as palmitic acid methyl ester 140).