Table 4.

Combinations of genetic perturbations suggested by OptForce procedure for triacylglycerol production under nitrogen limitation.

Intervention a		WT	Mutant strains
			k = 2	k = 3									k = 4
			1	2	3	4	5	6	7	8	9	10	11	12	13	14	15
↑ DGAT_rm	P (Rt)		X	X	X	X	X	X	X
↑ ACCOAC_c	P (Rt)									X
↑ ACOATA_c	P (O)										X					X	X
↑ ACOADS180_rm	P (Rt)											X	X	X	X
↑ ME2_c	C (Rt)			X									X
↑ G6PDH2i_c	C				X									X		X
↑ GLYCDy_c	C					X									X		X
↓ FECOOR_m	R						X
↓ ACONTa_m	R							X
↓ AKGDa_m	R								X
Δ beta-Oxidation b	D (O)									X	X	X	X	X	X	X	X
Δ Lipid degradation c	D (O)		X	X	X	X	X	X	X	X	X	X	X	X	X	X	X
TAG production flux (mmol/gDW/hr)		0.185	0.567	0.570	0.569	0.569	0.568	0.572	0.572	0.565	0.565	0.566	0.569	0.569	0.568	0.568	0.567

^aReaction abbreviations are listed in the table and annotated in the text. Reactions are described in Supplementary Materials 1. These interventions are classified into types of strategies: P – “push-pull”, C – cofactor regeneration, R – respiratory disruption, D – degradation knockout. Successful implementations in R. toruloides (Rt) (Zhang et al., 2016b, 2016a) or other oleaginous yeast (O) (Blazeck et al., 2014; Tamano et al., 2013) are reported.

^bbeta-Oxidation knockout was applied manually prior to finding FORCE set for strains #8-15 in order to block fatty acid degradation. The first step for beta-oxidation in peroxisome and mitochondria were knocked out, which are fatty acyl-CoA oxidase (encoded by POX1, rt4374, and rt4384) and dehydrogenase (encoded by rt5702, rt4202, and rt1644), respectively.

^cLipid degradation knockout was applied to all designs to produce TAG, including TAG lipase (encoded by TGL2, TGL5, and ATG15) and monoacylglycerol lipase (YJU3).