Table 1 List of plasmids and strains used in this study

Plasmids

BB1_12_pDAS1

P_DAS1, KanR⁺

[57]

BB1_12_pDAS2

P_DAS2, KanR⁺

BB1_12_SHB17

P_SHB17, KanR⁺

BB1_23

Ø, KanR⁺

BB1_34_RPS3tt

_RPS3tt, KanR⁺

BB1_34_ScCYC1tt

_ScCYC1tt, KanR⁺

BB2_BC

Ø, AmpR⁺

BB1_23_AAT2

AAT2⁺, K. phaffii gene encoding cytosolic aspartate aminotransferase Aat2

This work

BB1_23_SpeAspDH

aspDH⁺, S. proteamaculans codon-optimized gene encoding aspartate dehydrogenase AspDH

BB1_23_PYC2

PYC2⁺, K. phaffii codon-optimized gene encoding pyruvate carboxylase Pyc2

BB1_23_JEN1

JEN1⁺, K. phaffii gene encoding putative lactate transporter Jen1

BB1_23_ScESBP6

ESBP6⁺, S. cerevisiae gene encoding monocarboxylate permease Esbp6

BB1_23_PseFDH(V9)

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

[39]

BB2_BC_AAT2

P_DAS1-AAT2-RPS3tt

This work

BB2_BC_SpeAspDH

P_DAS1-AspDH_Spe-RPS3tt

BB2_BC_PYC2

P_SHB17-PYC2-RPS3tt

BB2_BC_JEN1

P_DAS2-JEN1-ScCYC1tt

BB2_BC_ScESBP6

P_DAS1-ESBP6_Sc-ScCYC1tt

BB2_BC_PseFDH(V9)

P_FDH1-FDH(V9)_Pse-TDH3tt

[39]

BB3nK_ext_AD

Ø, KanR⁺

[52]

BB3nK_ext_AD_AAT2

5’-HR_P_DAS1-AAT2-RPS3tt_3’-HR(pENO1^UP)

This work

BB3nK_ext_AD_SpeAspDH

5’-HR_P_DAS1-AspDH_Spe-RPS3tt_3’-HR(pENO1^UP)

BB3nK_ext_AD_PYC2

5’-HR_P_SHB17-PYC2-RPS3tt_3’-HR(AOX1tt_DOWN)

BB3nK_ext_AD_JEN1

5’-HR_P_DAS2-JEN1-ScCYC1tt_3’-HR(GUT1tt_DOWN)

5’-HR_P_DAS2-JEN1-ScCYC1tt_3’-HR(pOPT1-1^UP)

BB3nK_ext_AD_ScESBP6

5’-HR_P_DAS1-ESBP6_Sc-ScCYC1tt_3’-HR(GUT1tt_DOWN)

5’-HR_P_DAS1-ESBP6_Sc-ScCYC1tt_3’-HR(pENO1^UP)

BB3nK_ext_AD_PseFDH(V9)

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

[39]

BB3cN_pGAP_23*_pLAT1_Cas9

P_GAP_ Ø_P_LAT1_Cas9, NrsR⁺

[52]

BB3cN_pGAP_gRNA1(pENO1^UP) _pLAT1_Cas9

P_GAP_ gRNA1(P_ENO1^UP)_P_LAT1_Cas9, NrsR⁺

This work

BB3cN_pGAP_gRNA2(pENO1^UP) _pLAT1_Cas9

P_GAP_ gRNA2(P_ENO1^UP)_P_LAT1_Cas9, NrsR⁺

BB3cN_pGAP_gRNA1(AOX1tt_DOWN) _pLAT1_Cas9

P_GAP_ gRNA1(AOX1tt_DOWN)_P_LAT1_Cas9, NrsR⁺

BB3cN_pGAP_gRNA1(GUT1tt_DOWN) _pLAT1_Cas9

P_GAP_ gRNA1(GUT1tt_DOWN)_P_LAT1_Cas9, NrsR⁺

BB3cN_pGAP_gRNA2(GUT1tt_DOWN) _pLAT1_Cas9

P_GAP_ gRNA2(GUT1tt_DOWN)_P_LAT1_Cas9, NrsR⁺

BB3cN_pGAP_gRNA1(pOPT1-1^UP) _pLAT1_Cas9

P_GAP_ gRNA1(P_OPT1−1^UP)_P_LAT1_Cas9, NrsR⁺

BB3cN_pGAP_gRNA2(ΔFDH1) _pLAT1_Cas9

P_GAP_gRNA2(ΔFDH1)_P_LAT1_Cas9, NrsR⁺

BB3cN_pGAP_gRNA1(pAOX1^UP)

_pLAT1_Cas9

P_GAP_gRNA1(P_AOX1^UP)_P_LAT1_Cas9, NrsR⁺

[39]

K. phaffii strains

PpCβ21

2x(P_AOX1_PAND_Tc) + P_FDH1_BAPAT_Bc + P_POR1_YDFG_Ec

[39]

PpCβ21-P

PpCβ21 + P_FDH1_FDH(V9)_Pse

PpCβ21-A

PpCβ21 + P_DAS1_AAT2

This work

PpCβ21-D

PpCβ21 + P_DAS1_AspDH_Spe

PpCβ21-Y

PpCβ21 + P_SHB17_PYC2

PpCβ21-YA

PpCβ21-Y + P_DAS1_AAT2

PpCβ21-YAP

PpCβ21-YA + P_FDH1_FDH(V9)_Pse

PpCβ21-PE

PpCβ21-P + P_DAS1_ESBP6_Sc

PpCβ21-PJ

PpCβ21-P + P_DAS2_JEN1

PpCβ21-PEJ

PpCβ21-PE + P_DAS2_JEN1

PpCβ21-PEJY

PpCβ21-PEJ + P_SHB17_PYC2

PpCβ21-PEJΔfdh1

PpCβ21-PEJ + FDH1⁻

PpCβ21-PEJYΔfdh1

PpCβ21-PEJY + FDH1⁻