Engineering xylose metabolism in thraustochytrid T18

Table 2 Gene copy number of wild-type and transformants and protein activity on xylose and xylulose

Strain	Gene copy number^a			% sugar used/μg/μL sugar left^b		μg/μL xylulose-5-phosphate^c
Strain	ble ^d	xi	aph7	Xylose	Xylulose	μg/μL xylulose-5-phosphate^c
WT	–	2.0	–	− 0.21 ± 0.28/0.55 ± 0.002	1.59 ± 0.58/0.62 ± 0.004	0.000 ± 0.004
XI 16	16.3	14.6	–	0.26 ± 0.29/0.55 ± 0.002	12.9 ± 0.26/0.55 ± 0.002	0.000 ± 0.001
XI-XK 1	22.7	23.2	1.8	10.2 ± 0.21/0.50 ± 0.002	42.5 ± 0.38/0.36 ± 0.002	0.035 ± 0.001
XI-XK 3	1.6	3.8	2.0	0.67 ± 0.13/0.55 ± 0.001	8.88 ± 0.67/0.58 ± 0.004	0.001 ± 0.004
XI-XK 7	26.1	26.6	23.2	26.2 ± 0.32/0.41 ± 0.002	92.1 ± 0.47/0.05 ± 0.003	0.404 ± 0.003
XB	23.6	2.0	–	0.14 ± 0.34/0.55 ± 0.002	97.8 ± 3.9/0.01 ± 0.025	0.619 ± 0.026

N.D. not determined
^aGene copy numbers were determined by qPCR
^bCell extracts were done with set amounts of xylose (0.55 μg/μL) and xylulose (0.63 μg/μL) and the amount of sugar detected was determined by HPLC. The amount of sugar converted or detected following incubation with 0.08 μg/μL total protein extract is shown
^cXylulose-5-phosphate produced with 0.08 μg/μL total protein was calculated by deducting the total amount of xylose and xylulose detected in these reactions from the known starting amount of xylulose
^dble values in the XB transformant indicate the gene copies of ble-2A-xylB, whereas, in the XI and XI-XK transformants, it indicates gene copies of ble-2A-his-xi

ISSN: 2731-3654