Table 3 List of plasmids and strains used in this study

Plasmids

 BB1_12_pAOX1

P_AOX1, KanR⁺

[5]

 BB1_12_pFDH1

P_FDH1, KanR⁺

 BB1_12_pPOR1

P_POR1, KanR⁺

 BB1_12_pPDC1

P_PDC1, KanR⁺

 BB1_23

Ø, KanR⁺

 BB1_34_RPS3tt

_RPS3tt, KanR⁺

 BB1_34_ScCYC1tt

_ScCYC1tt, KanR⁺

 BB1_34_TDH3tt

_TDH3tt, KanR⁺

 BB2_BC

Ø, AmpR⁺

 pCC1-4k_TcPAND

panD⁺, T. castaneum gene encoding for PAND_Tc enzyme

GenScript Biotech

 pCC1-4k_BcBAPAT

yhxA⁺, B. cereus gene encoding for BAPAT_Bc

 BB1_23_EcYDFG

ydfG⁺, E. coli str. K-12 gene encoding for YDFG_Ec

 BB1_23_PseFDH(V9)

fdh⁺, Pseudomonas sp. (strain 101) gene encoding for FDH_Pse(V9)_{(A199G/D222Q/S381V/C256A/H380K)}

 BB2_BC_TcPAND

P_AOX1-PAND_Tc-RPS3tt

This work

 BB2_BC_BcBAPAT

P_FDH1-BAPAT_Bc-ScCYC1tt

 BB2_BC_EcYDFG

P_POR1-YDFG_Ec-TDH3tt

P_PDC1-YDFG_Ec-TDH3tt

 BB2_BC_PseFDH(V9)

P_FDH1-FDH(V9)_Pse-TDH3tt

 BB3nK_ext_AD

Ø, KanR⁺

[6]

 BB3nK_ext_AD_TcPAND

5′-HR_P_AOX1-PAND_Tc-RPS3tt_3′-HR(RGI2)

This work

5′-HR_P_AOX1-PAND_Tc-RPS3tt_3′-HR(pGAP^UP)

 BB3nK_ext_AD_BcBAPAT

5′-HR_P_FDH1-BAPAT_Bc-ScCYC1tt_3′-HR(pTEF1-α^UP)

 BB3nK_ext_AD_EcYDFG

5′-HR_P_POR1-YDFG_Ec-TDH3tt_3′-HR(pFLD1^UP)

5′-HR_P_PDC1-YDFG_Ec-TDH3tt_3′-HR(pFLD1^UP)

 BB3nK_ext_AD_PseFDH(V9)

5′-HR_P_FDH1-FDH(V9)_Pse-TDH3tt_3′-HR(pAOX1^UP)

 BB3cN_pGAP_23*_pLAT1_Cas9

P_GAP_Ø_P_LAT1_Cas9, NrsR⁺

[6]

 BB3cK_pGAP_23*_pLAT1_Cas9

P_GAP_Ø_P_LAT1_Cas9, KanR⁺

 BB3cH_pGAP_23*_pLAT1_Cas9

P_GAP_Ø_P_LAT1_Cas9, HygR⁺

 BB3cK_pGAP_gRNA1(RGI2)_pLAT1_Cas9

P_GAP_gRNA1(RGI2)_P_LAT1_Cas9, KanR⁺

This work

 BB3cH_pGAP_gRNA1(pGAP^UP)_pLAT1_Cas9

P_GAP_gRNA1(P_GAP^UP)_P_LAT1_Cas9, HygR⁺

 BB3cK_pGAP_gRNA2(pTEF1-α^UP)_pLAT1_Cas9

P_GAP_gRNA2(P_TEF1-α^UP)_P_LAT1_Cas9, KanR⁺

 BB3cN_pGAP_gRNA1(pFLD1^UP)_pLAT1_Cas9

P_GAP_gRNA1(P_FLD1^UP)_P_LAT1_Cas9, NrsR⁺

 BB3cN_pGAP_gRNA1(pAOX1^UP)_pLAT1_Cas9

P_GAP_gRNA1(P_AOX1^UP)_P_LAT1_Cas9, NrsR⁺

K. phaffii strains

 CBS7435

Wild-type

CBS, Centraalbureau voor Schimmelcultures

 PpCβ10

P_AOX1_PAND_Tc + P_FDH1_BAPAT_Bc + P_POR1_YDFG_Ec

This work

 PpCβ20

P_AOX1_PAND_Tc + P_FDH1_BAPAT_Bc + P_PDC1_YDFG_Ec

 PpCβ11

2x(P_AOX1_PAND_Tc) + P_FDH1_BAPAT_Bc + P_POR1_YDFG_Ec

 PpCβ21

2x(P_AOX1_PAND_Tc) + P_FDH1_BAPAT_Bc + P_PDC1_YDFG_Ec

 PpCβ20-P

PpCβ20 + P_FDH1_FDH(V9)_Pse

 PpCβ21-P

PpCβ21 + P_FDH1_FDH(V9)_Pse