Screening of endogenous strong promoters for enhanced production of medium-chain-length polyhydroxyalkanoates in Pseudomonas mendocina NK-01


Table S1

The top 30 genes ranked by transcriptional level based on their RPKM values in RNA-seq analysis Table S2 The molecular weights of mcl-PHAs synthesized by P. mendocina NKU and its mutants Table S3 The monomer composition ratios of mcl-PHA synthesized by P. mendocina 

NKU and its mutants

Table S4 Primers used in this study 
gfp-P9-F P16-F P16-R gfp-16-F P17-F P17-R gfp-17-F P18-F P18-R gfp-18-F P20-F P20-R gfp-20-F AACCCCGCTGAATTCAGGAGGCGAATTCATGAGTAAAGGAGAAGAAC TATAGGGCGAATTGGAGCTCGGTACCATCCGAGGGATACGGGCCTTGTC ATGAATTCGCCTCCTGAATTCGAGCTTGATCGCGGATTAACAC ATGAATTCGCCTCCTGAATTCGAGCTTGATCGCGGATTAACAC TATAGGGCGAATTGGAGCTCGGTACCGCCAGATCGACTGAAGGCACAAG ATGAATTCGCCTCCTGAATTCGTTGCCTTGTGTAAGAGACG CAAGGCAACGAATTCAGGAGGCGAATTCATGAGTAAAGGAGAAGAAC TATAGGGCGAATTGGAGCTCGGTACC AGCTTCAAGCGGCAAGCCAC ATGAATTCGCCTCCTGAATTCGACAGTTAGCGGAAACGCCCAC CTAACTGTCGAATTCAGGAGGCGAATTCATGAGTAAAGGAGAAGAAC TATAGGGCGAATTGGAGCTCCAGGACGAACCTAGCTGAAC ATGAATTCGCCTCCTGAATTCGAATTCAGAGGTATCCTGATCCATTAG GATACCTCTGAATTCAGGAGGCGAATTCATGAGTAAAGGAGAAGAAC P23-R gfp-P23-F P25-F P25-R gfp-P25-F P29-F P29-R gfp-P29-F gfp-R gfp-lac-F gfp-lac-R

Fig. S1

 S1 Fig. S1 Cloned promoter sequences and predicted promoter sequences 


Fig. S2

 S2 Fig. S2The growth curves of all the constructed strains for promoter characterization 


Fig. S1

 S1 Fig. S1 Cloned promoter sequences and predicted promoter sequences. Each cloned 


Fig. S2

 S2 Fig. S2 The growth curves of all the constructed strains for promoter characterization. Data represent the mean values ± standard deviations of triplicate measurements from three independent experiments. 


Fig. S3

 S3 Fig. S3 Transcriptional levels of the five strong promoters and lac promoter in different growth phases. The relative transcriptional value of each promoter in early log phase was set as 1. Data represent the mean values ± standard deviations of triplicate measurements from three independent experiments. 


Fig. S4

 S4 Fig. S4 GC-MS analysis for the monomer compositions of mcl-PHAs synthesized by 


Fig. S5

 S5 Fig. S5 The molecular structure of mcl-PHA produced by P. mendocina NK-01 




  




  




  


Table S1

 S1 The top 30 genes ranked by transcriptional level based on their RPKM values in RNA-seq analysis 


	Gene ID	Productions	Promoters	RPKM
	MDS_4427	hypothetical protein	P1	36681.95
	MDS_r003	16S ribosomal RNA	P2	33511.23
	MDS_1604	hypothetical protein	P3	29531.20
	MDS_2118	hypothetical protein	P4	24593.43
	MDS_1524	anti sigma-E protein,RseA	P5	24300.80
	MDS_0756	hypothetical protein	P6	24066.04
	MDS_2424	general stress protein	P7	19061.06
	MDS_3781	hypothetical protein	P8	17212.90
	MDS_3450	hypothetical protein	P9	16182.31
	MDS_3307	hypothetical protein	P10	15688.80
	MDS_4616	hypothetical protein	P11	14094.26
	MDS_1176	UspA domain-containing protein	P12	13610.24
	MDS_1400	hypothetical protein	P13	13273.27
	MDS_2117	hypothetical protein	P14	10945.60
	MDS_3417	glyoxalase/bleomycin resistance protein/dioxygenase	P15	9162.63
	MDS_2531	alcohol dehydrogenase	P16	8949.33
	MDS_0957	ribosomal protein S30P / sigma 54 modulation protein	P17	8412.46
	MDS_0563	poly(hydroxyalkanoate) granule-associated protein	P18	8222.26
	MDS_2561	DNA-binding transcriptional activator	P19	7903.00
	MDS_1497	17 kDa surface antigen/outer membrane lipoprotein	P20	7267.13
	MDS_3574	PfpI family intracellular peptidase	P21	7239.34
	MDS_4560	hypothetical protein	P22	7213.88
	MDS_1161	arginine/ornithine antiporter	P23	6935.83
	MDS_2084	hypothetical protein	P24	6818.40
	MDS_4257	outer membrane protein W	P25	6637.68
	MDS_1159	ornithine carbamoyltransferase	P26	6455.59
	MDS_2628	OmpF family protein	P27	5967.23
	MDS_0564	poly(hydroxyalkanoate) granule-associated protein	P28	5905.14
	MDS_3908	transport-associated protein	P29	5888.12
	MDS_2110	transglutaminase domain-containing protein	P30	5796.17

Table S2

 S2 The molecular weights of mcl-PHAs synthesized by P. mendocina NKU and 


	its mutants			
	Strains	Mw/ ×10 4 Da Mn/ ×10 4 Da	Mw/Mn
	NKU	25.2	14.3	1.76
	NKU-∆phaZ	22.5	15.8	1.42
	NKU-∆phaZ-4C1	18.8	13.3	1.41
	NKU-∆phaZ-6C1	29.1	15.1	1.93
	NKU-∆phaZ-16C1	17.5	11.7	1.50

Table S3

 S3 The monomer composition ratios of mcl-PHA synthesized by P. mendocina 


	Table S4 Primers used in this study		
	Primers	Description			Source or literature
	P4-F	TATAGGGCGAATTGGAGCTCGGTACCGAGGTTTCCTGTGGTGAGCA	This work
	P4-R	NKU and its mutants ATGAATTCGCCTCCTGAATTCGTGGGCGTGTCAGCTTGG		This work
	gfp-P4-F	CACGCCCACGAATTCAGGAGGCGAATTCATGAGTAAAGGAGAAGAAC	This work
	P6-F	TATAGGGCGAATTGGAGCTCGGTACCGGCTGAAGCGGCCTCCT	This work
	P6-R	ATGAATTCGCCTCCTGAATTCGTTGGGGAGTAATAAGTGGCTTC	This work
	gfp-P6-F	Monomer composition (%) CTCCCCAACGAATTCAGGAGGCGAATTCATGAGTAAAGGAGAAGAAC	This work
	P9-F	Strains TATAGGGCGAATTGGAGCTCGGTACCCCTGCTGCGCCTTTCACAAGAC C8 C10	C12	This work
	P9-R	ATGAATTCGCCTCCTGAATTCAGCGGGGTTGTGGTTGGGTATG	This work
		NKU	17.87	77.84	4.29
		NKU-∆phaZ	16.27	79.35	4.38
		NKU-∆phaZ-4C1	19.38	74.39	6.23
		NKU-∆phaZ-6C1	22.13	73.41	4.46
		NKU-∆phaZ-16C1	15.11	79.86	5.03

This work This work This work This work This work This work This work This work This work This work This work This work This work P23-F This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work 7 gfp-QF TGCACTACTGGAAAACTACC This work gfp-QR ACTCGATGCGATTAACAAGG This work phaC1-QF GGCACTCAAACAACCCTTCC This work phaC1-QR TCACCGTCTTCGGGCTTTAG This work phaZ-QF TGGTGCCACTGCTGATCTTC This work phaZ-QR TGGCCGTAGTCGAGGTAATC This work phaC2-QF GCACCTCAATCCCTTCTATC This work phaC2-QR TGTTGAACAGCTCCTTTACC This work 16S-QF TCACGATTACTAGCGATTCC This work 16S-QR AACCCTTGTCCTTAGTTACC This work
Fengjie Zhao, Xiangsheng Liu, Annie Kong, Yuxin Zhao, Xu Fan, Ting Ma, Weixia Gao, Shufang Wang, Chao Yang
Screening of endogenous strong promoters for enhanced production of medium-chain-length polyhydroxyalkanoates in Pseudomonas mendocina NK-01