微生物学报

Strains	Genotype/Description	Reference/Source
Serratia marcescens BoR121	Wild-type, prodigiosin producer	This work
Escherichia coli
DH5α	F^–, φ80lacZΔM15, Δ(lacZYA-argF) U169, recA1, endA1, hsdR17 (rK^–, mK⁺), phoA, supE44, λ^–, thi-1, gyrA96, relA1	Thermo Fisher Scientific
JM109	endA1, recA1, gyrA96, thi, hsdR17 (r_k^–, m_k⁺), relA1, supE44, Δ(lac-proAB), [F′ traD36, proAB, lacI^qZΔM15]	Thermo Fisher Scientific
BL21(DE3)	fhuA2, ompT, gal (λ DE3), [dcm] ∆hsdS, λ DE3=λ sBamH Io ∆EcoR Ⅰ-B int-(lacI-PlacUV5-T7 gene1) i21 ∆nin5	New England Biolabs
BAP1	BL21(DE3), ΔprpRBCD-T7prom-sfp, T7prom-prpE	Kerafast
BW25113/pIJ790	K-12 derivative, ΔaraBAD, ΔrhaBAD/pIJ790	[26]
GB05-dir	Red/ET mediated linear-linear recombineering	[27-28]
DH5α-pig	E. coli DH5α containing p15A: : pig	This work
JM109-pig	E. coli JM109 containing p15A: : pig	This work
BL21-pig	E. coli BL21(DE3) containing p15A: : pig	This work
BAP1-pig	E. coli BAP1 containing p15A: : pig	This work
BAP1-T7-pig	E. coli BAP1 containing pET28a: : T7 pig	This work
BAP1-rpoD-pig	E. coli BAP1 containing pET28a: : P_rpoD pig	This work
BAP1-rpoS-pig	E. coli BAP1 containing pET28a: : P_rpoS pig	This work
BAP1-oxb20-pig	E. coli BAP1 containing pET28a: : P_oxb20 pig	This work

Strains	Genotype/Description	Reference/Source
Serratia marcescens BoR121	Wild-type, prodigiosin producer	This work
Escherichia coli
DH5α	F^–, φ80lacZΔM15, Δ(lacZYA-argF) U169, recA1, endA1, hsdR17 (rK^–, mK⁺), phoA, supE44, λ^–, thi-1, gyrA96, relA1	Thermo Fisher Scientific
JM109	endA1, recA1, gyrA96, thi, hsdR17 (r_k^–, m_k⁺), relA1, supE44, Δ(lac-proAB), [F′ traD36, proAB, lacI^qZΔM15]	Thermo Fisher Scientific
BL21(DE3)	fhuA2, ompT, gal (λ DE3), [dcm] ∆hsdS, λ DE3=λ sBamH Io ∆EcoR Ⅰ-B int-(lacI-PlacUV5-T7 gene1) i21 ∆nin5	New England Biolabs
BAP1	BL21(DE3), ΔprpRBCD-T7prom-sfp, T7prom-prpE	Kerafast
BW25113/pIJ790	K-12 derivative, ΔaraBAD, ΔrhaBAD/pIJ790	[26]
GB05-dir	Red/ET mediated linear-linear recombineering	[27-28]
DH5α-pig	E. coli DH5α containing p15A: : pig	This work
JM109-pig	E. coli JM109 containing p15A: : pig	This work
BL21-pig	E. coli BL21(DE3) containing p15A: : pig	This work
BAP1-pig	E. coli BAP1 containing p15A: : pig	This work
BAP1-T7-pig	E. coli BAP1 containing pET28a: : T7 pig	This work
BAP1-rpoD-pig	E. coli BAP1 containing pET28a: : P_rpoD pig	This work
BAP1-rpoS-pig	E. coli BAP1 containing pET28a: : P_rpoS pig	This work
BAP1-oxb20-pig	E. coli BAP1 containing pET28a: : P_oxb20 pig	This work

Plasmids	Relevant characteristics	Reference/Source
p15A-cm-TetR-TetO	ori (p15A), Cm^R	[27]
pET28a	ori (pBR322), T7, His6·tag, Kan^R	Novagen
p15A: : pig	A derivative of p15A containing the prodigiosin biosynthetic gene cluster, Cm^R	This work
pET28a: : T7 pigUpDn	pET28a containing T7 promoter, 500 bp of pigA1 and 530 bp of copA	This work
pET28a: : P_rpoD pigUpDn	pET28a containing P_rpoD, 500 bp of pigA3 and 530 bp of copA	This work
pET28a: : P_oxb20 pigUpDn	pET28a containing P_oxb20, 500 bp of pigA3 and 530 bp of copA	This work
pET28a: : P_rpoS pigUpDn	pET28a containing P_rpoS, 500 bp of pigA2 and 530 bp of copA	This work
pET28a: : T7 pig	A derivative of pET28a containing the prodigiosin biosynthetic gene cluster driven by T7 promoter	This work
pET28a: : P_oxb20 pig	A derivative of pET28a containing the prodigiosin biosynthetic gene cluster driven by oxb20 promoter	This work
pET28a: : P_rpoD pig	A derivative of pET28a containing the prodigiosin biosynthetic gene cluster driven by rpoD promoter	This work
pET28a: : P_rpoS pig	A derivative of pET28a containing the prodigiosin biosynthetic gene cluster driven by rpoS promoter	This work

Plasmids	Relevant characteristics	Reference/Source
p15A-cm-TetR-TetO	ori (p15A), Cm^R	[27]
pET28a	ori (pBR322), T7, His6·tag, Kan^R	Novagen
p15A: : pig	A derivative of p15A containing the prodigiosin biosynthetic gene cluster, Cm^R	This work
pET28a: : T7 pigUpDn	pET28a containing T7 promoter, 500 bp of pigA1 and 530 bp of copA	This work
pET28a: : P_rpoD pigUpDn	pET28a containing P_rpoD, 500 bp of pigA3 and 530 bp of copA	This work
pET28a: : P_oxb20 pigUpDn	pET28a containing P_oxb20, 500 bp of pigA3 and 530 bp of copA	This work
pET28a: : P_rpoS pigUpDn	pET28a containing P_rpoS, 500 bp of pigA2 and 530 bp of copA	This work
pET28a: : T7 pig	A derivative of pET28a containing the prodigiosin biosynthetic gene cluster driven by T7 promoter	This work
pET28a: : P_oxb20 pig	A derivative of pET28a containing the prodigiosin biosynthetic gene cluster driven by oxb20 promoter	This work
pET28a: : P_rpoD pig	A derivative of pET28a containing the prodigiosin biosynthetic gene cluster driven by rpoD promoter	This work
pET28a: : P_rpoS pig	A derivative of pET28a containing the prodigiosin biosynthetic gene cluster driven by rpoS promoter	This work

Primers	Sequences (5′→3′)	Purpose
Underlined sequences for restriction enzyme recognition sites, and lowercase letters for overlapping between DNA sequences.
27F	AGAGTTTGATCCTGGCTCAG	PCR amplification of 16S rRNA gene
1492R	GGTTACCTTGTTACGCTT	PCR amplification of 16S rRNA gene
pig-HA-p15A-1-F	gccgggcgagcgtcggctccatctggtgggcagcatccaAGATCCGAAAACCCCAAGTT	p15A vector fragment carrying the 80 bp target gene cluster homology arm
pig-HA-p15A-1-R	atgggaataaaagtaagcatgcaaacctcctgtcagacaAGATCCTTTCTCCTCTTTAG
pig-HA-p15A-2-F	tcgcccaaaagttacgcttatccggcgctggacatcactctGCCGGGCGAGCGTCGGCT
pig-HA-p15A-2-R	tgacgccggcgaacaccacaatcagcgcgacgacgatcacgATGGGAATAAAAGTAAGC
oxb20-F	AATTAGATCTCTATTTACAAGAGGGGGGCG	PCR 706 bp of oxb20
oxb20-R	GCCGGGTAATACCGGATAGT	PCR 706 bp of oxb20
rpoD-F	AATTAGATCTACGCACGACCATGCGTATAC	PCR 600 bp of rpoD
rpoD-R	tgataagttaaaatccatAAGACGGTATCCACACTTAT	PCR 600 bp of rpoD
rpoS-F	AATTAGATCTCTTTAGTGAAGGGTGAGCTGGC	PCR 1 367 bp of rpoS
rpoS-R	AAGGTGGCTCCTACCCGTGA	PCR 1 367 bp of rpoS
pig Up-F1	AATTCCATGGATGGATTTTAACTTATCAAGTGGGC	PCR 500 bp of pigA1
pig Up-F2	ATGGATTTTAACTTATCAAGTGGGC	PCR 500 bp of pigA2
pig Up-F3	tatccggtattacccggcATGGATTTTAACTTATCAAGTGGG	PCR 500 bp of pigA3
pig Up-R	TTAAGCTAGCATGTCGGCAATCGGCGCGTT	PCR 500 bp of pigA1−A3
pig Dn-F	AATTGCTAGCCCCCAACGCGGCAGCGCGGTT	PCR 530 bp of copA
pig Dn-R	AATTGAATTCGGATCCCTTTGCGTTGTCGATCTCG	PCR 530 bp of copA

Primers	Sequences (5′→3′)	Purpose
Underlined sequences for restriction enzyme recognition sites, and lowercase letters for overlapping between DNA sequences.
27F	AGAGTTTGATCCTGGCTCAG	PCR amplification of 16S rRNA gene
1492R	GGTTACCTTGTTACGCTT	PCR amplification of 16S rRNA gene
pig-HA-p15A-1-F	gccgggcgagcgtcggctccatctggtgggcagcatccaAGATCCGAAAACCCCAAGTT	p15A vector fragment carrying the 80 bp target gene cluster homology arm
pig-HA-p15A-1-R	atgggaataaaagtaagcatgcaaacctcctgtcagacaAGATCCTTTCTCCTCTTTAG
pig-HA-p15A-2-F	tcgcccaaaagttacgcttatccggcgctggacatcactctGCCGGGCGAGCGTCGGCT
pig-HA-p15A-2-R	tgacgccggcgaacaccacaatcagcgcgacgacgatcacgATGGGAATAAAAGTAAGC
oxb20-F	AATTAGATCTCTATTTACAAGAGGGGGGCG	PCR 706 bp of oxb20
oxb20-R	GCCGGGTAATACCGGATAGT	PCR 706 bp of oxb20
rpoD-F	AATTAGATCTACGCACGACCATGCGTATAC	PCR 600 bp of rpoD
rpoD-R	tgataagttaaaatccatAAGACGGTATCCACACTTAT	PCR 600 bp of rpoD
rpoS-F	AATTAGATCTCTTTAGTGAAGGGTGAGCTGGC	PCR 1 367 bp of rpoS
rpoS-R	AAGGTGGCTCCTACCCGTGA	PCR 1 367 bp of rpoS
pig Up-F1	AATTCCATGGATGGATTTTAACTTATCAAGTGGGC	PCR 500 bp of pigA1
pig Up-F2	ATGGATTTTAACTTATCAAGTGGGC	PCR 500 bp of pigA2
pig Up-F3	tatccggtattacccggcATGGATTTTAACTTATCAAGTGGG	PCR 500 bp of pigA3
pig Up-R	TTAAGCTAGCATGTCGGCAATCGGCGCGTT	PCR 500 bp of pigA1−A3
pig Dn-F	AATTGCTAGCCCCCAACGCGGCAGCGCGGTT	PCR 530 bp of copA
pig Dn-R	AATTGAATTCGGATCCCTTTGCGTTGTCGATCTCG	PCR 530 bp of copA

Feature	Chromosome characteristics
Chromosome size (bp)	5 228 182
G+C content (%)	59.51
Predicted genes	4 996
rRNA operons	22
tRNA genes	92
sRNA genes	112
Genes assigned to COG	4 502
Genes assigned to GO	3 136
Genes assigned to KEGG	3 098
CRISPR repeat regions	4
Prophages	3
Simple tandem repeats	26
Interspersed repeats	72
Genomic islands	8
Carbohydrate-active enzymes	138
Transposable element	9
Antibiotic resistance genes	322
Virulence factors	656
Secondary metabolite gene clusters	11

Feature	Chromosome characteristics
Chromosome size (bp)	5 228 182
G+C content (%)	59.51
Predicted genes	4 996
rRNA operons	22
tRNA genes	92
sRNA genes	112
Genes assigned to COG	4 502
Genes assigned to GO	3 136
Genes assigned to KEGG	3 098
CRISPR repeat regions	4
Prophages	3
Simple tandem repeats	26
Interspersed repeats	72
Genomic islands	8
Carbohydrate-active enzymes	138
Transposable element	9
Antibiotic resistance genes	322
Virulence factors	656
Secondary metabolite gene clusters	11

Cluster	Type	From	To	Most similar known biosynthetic gene cluster
1	Hserlactone	23 358	44 032	Unknown
2	NRPS	278 246	342 472	Unknown
3	Betalactone	747 506	773 174	Unknown
4	NRPS-PKS	1 174 609	1 195 556	Prodigiosin
5	Thiopeptide	1 797 266	1 823 709	Unknown
6	Other	2 183 729	2 205 826	Yersinopine
7	Other	2 467 098	2 487 376	Unknown
8	NRPS-PKS	2 514 439	2 584 366	Unknown
9	NRPS	3 218 021	3 294 757	Unknown
10	Thiopeptide	3 840 325	3 887 793	Unknown
11	NRPS	4 687 524	4 731 463	Unknown

Cluster	Type	From	To	Most similar known biosynthetic gene cluster
1	Hserlactone	23 358	44 032	Unknown
2	NRPS	278 246	342 472	Unknown
3	Betalactone	747 506	773 174	Unknown
4	NRPS-PKS	1 174 609	1 195 556	Prodigiosin
5	Thiopeptide	1 797 266	1 823 709	Unknown
6	Other	2 183 729	2 205 826	Yersinopine
7	Other	2 467 098	2 487 376	Unknown
8	NRPS-PKS	2 514 439	2 584 366	Unknown
9	NRPS	3 218 021	3 294 757	Unknown
10	Thiopeptide	3 840 325	3 887 793	Unknown
11	NRPS	4 687 524	4 731 463	Unknown

ORF	Number of amino acids	Proposed function	Protein homology	Amino acid identity (%)
pigA	385	l-prolyl-PCP dehydrogenase	Serratia marcescens acyl-CoA dehydrogenase family protein (MDV2100735.1)	383/385 (99)
pigB	670	Oxidoreductase	Serratia marcescens FAD-dependent oxidoreductase (HEJ0404376.1)	667/670 (99)
pigC	888	Prodigiosin synthesizing transferase	Serratia marcescens phosphoenolpyruvate synthase (MDR2288025.1)	884/888 (99)
pigD	867	Thiamine diphosphate dependent-3-acetyloctanal synthase	Serratia marcescens biosynthesis protein PigD (WP_197787955.1)	864/867 (99)
pigE	853	Aminotransferase	Serratia sp. JKS296 minotransferase class Ⅲ-fold pyridoxal phosphate-dependent enzyme (WP_097102633.1)	851/853 (99)
pigF	338	S-adenosyl-l-methionine-dependent methyl transferase	Serratia marcescens methyltransferase domain-containing protein (MBH2772015.1)	336/338 (99)
pigG	87	Acyl carrier protein	Serratia marcescens acyl carrier protein (WP_176063587.1)	86/87 (99)
pigH	648	4-hydroxy-2, 2′-bipyrrole-5-methanol synthase	Serratia sp. JKS296 aminotransferase class Ⅰ/Ⅱ-fold pyridoxal phosphate-dependent enzyme (WP_097102635.1)	643/648 (99)
pigI	491	l-proline-[l-prolyl-carrier protein] ligase	Serratia marcescens d-alanine-poly(phosphoribitol) ligase (AWO78120.1)	489/491 (99)
pigJ	762	Beta-ketoacyl synthase	Serratia marcescens polyketide synthase (WP_261420020.1)	758/762 (99)
pigK	104	Sterol 3-beta-glucosyltransferase	Serratia marcescens RedY (WP_164052918.1)	103/104 (99)
pigL	215	4′-phosphopantetheinyl transferase Sfp	Serratia marcescens 4′-phosphopantetheinyl transferase superfamily protein (WP_306376640.1)	212/215 (99)
pigM	352	Probable NAD(P)H nitroreductase	Serratia marcescens prodigiosin biosynthesis protein PigM (WP_229597835.1)	350/352 (99)
pigN	242	Delta (14)-sterol reductase	Serratia marcescens putative oxidoreductase (QWV59760.1)	240/242 (99)

ORF	Number of amino acids	Proposed function	Protein homology	Amino acid identity (%)
pigA	385	l-prolyl-PCP dehydrogenase	Serratia marcescens acyl-CoA dehydrogenase family protein (MDV2100735.1)	383/385 (99)
pigB	670	Oxidoreductase	Serratia marcescens FAD-dependent oxidoreductase (HEJ0404376.1)	667/670 (99)
pigC	888	Prodigiosin synthesizing transferase	Serratia marcescens phosphoenolpyruvate synthase (MDR2288025.1)	884/888 (99)
pigD	867	Thiamine diphosphate dependent-3-acetyloctanal synthase	Serratia marcescens biosynthesis protein PigD (WP_197787955.1)	864/867 (99)
pigE	853	Aminotransferase	Serratia sp. JKS296 minotransferase class Ⅲ-fold pyridoxal phosphate-dependent enzyme (WP_097102633.1)	851/853 (99)
pigF	338	S-adenosyl-l-methionine-dependent methyl transferase	Serratia marcescens methyltransferase domain-containing protein (MBH2772015.1)	336/338 (99)
pigG	87	Acyl carrier protein	Serratia marcescens acyl carrier protein (WP_176063587.1)	86/87 (99)
pigH	648	4-hydroxy-2, 2′-bipyrrole-5-methanol synthase	Serratia sp. JKS296 aminotransferase class Ⅰ/Ⅱ-fold pyridoxal phosphate-dependent enzyme (WP_097102635.1)	643/648 (99)
pigI	491	l-proline-[l-prolyl-carrier protein] ligase	Serratia marcescens d-alanine-poly(phosphoribitol) ligase (AWO78120.1)	489/491 (99)
pigJ	762	Beta-ketoacyl synthase	Serratia marcescens polyketide synthase (WP_261420020.1)	758/762 (99)
pigK	104	Sterol 3-beta-glucosyltransferase	Serratia marcescens RedY (WP_164052918.1)	103/104 (99)
pigL	215	4′-phosphopantetheinyl transferase Sfp	Serratia marcescens 4′-phosphopantetheinyl transferase superfamily protein (WP_306376640.1)	212/215 (99)
pigM	352	Probable NAD(P)H nitroreductase	Serratia marcescens prodigiosin biosynthesis protein PigM (WP_229597835.1)	350/352 (99)
pigN	242	Delta (14)-sterol reductase	Serratia marcescens putative oxidoreductase (QWV59760.1)	240/242 (99)

一株产灵菌红素黏质沙雷氏菌的分离鉴定及其基因组分析

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张雨欣 ¹ , 王美燕 ¹ , 李殿怡 ¹ , 司军 ³ , 卞小莹 ⁴ , 牛国清 ¹^,^* , 刘蓉 ¹^,²^,^*

微生物学报 | 研究报告 2024,64(11): 4290-4307

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微生物学报 | 研究报告 2024, 64(11): 4290-4307

一株产灵菌红素黏质沙雷氏菌的分离鉴定及其基因组分析

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张雨欣¹, 王美燕¹, 李殿怡¹, 司军³, 卞小莹⁴, 牛国清¹^,^*, 刘蓉¹^,²^,^*

作者信息

1 西南大学农学与生物科技学院, 重庆 400715

2 重庆中烟工业有限责任公司, 烟草资源科学利用重庆重点实验室, 重庆 400060

3 西南大学园艺园林学院, 长江上游农业生物安全与绿色生产教育部重点实验室, 重庆 400715

4 山东大学, 微生物技术国家重点实验室, 山东青岛 266237

Isolation, identification, and genomic analysis of a prodigiosin-producing strain of Serratia marcescens

Yuxin ZHANG¹, Meiyan WANG¹, Dianyi LI¹, Jun SI³, Xiaoying BIAN⁴, Guoqing NIU¹^,^*, Rong LIU¹^,²^,^*

Affiliations

1 College of Agronomy and Biotechnology, Southwest University, Chongqing 400715, China

2 Chongqing Key Laboratory of Scientific Utilization of Tobacco Resources, China Tobacco Chongqing Industrial Co., Ltd., Chongqing 400060, China

3 Key Laboratory of Agricultural Biosafety and Green Production of Upper Yangtze River, Ministry of Education, College of Horticulture and Landscape Architecture, Southwest University, Chongqing 400715, China

4 State Key Laboratory of Microbial Technology, Shandong University, Qingdao 266237, Shandong, China

出版时间: 2024-07-06 doi: 10.13343/j.cnki.wsxb.20240303

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摘要

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【目的】对甘蓝根际微生物进行分离，从中选择具有优良性状的菌株，对其次级代谢产物合成能力进行评估，为甘蓝根际微生物资源的发掘利用奠定基础。【方法】采集重庆市武隆区仙女山镇仙女山村甘蓝根，分离培养根际微生物菌株，从中选择一株黏质沙雷氏菌(Serratia marcescens)，利用PacBio RS Ⅱ平台和Illumina HiSeq平台完成全基因组测序，通过antiSMASH分析评估其合成次级代谢产物的潜力，采用Red/ET重组工程技术克隆目的基因簇并进行异源表达。【结果】从甘蓝根际分离鉴定到一株产灵菌红素的黏质沙雷氏菌，对其进行基因组测序及分析发现，基因组含有11个次级代谢产物生物合成基因簇，其中9个基因簇与已知化合物编码基因簇相似性较低，说明该菌具有产生多种新型次级代谢产物的潜力；HPLC分析和高分辨质谱检测鉴定了灵菌红素；克隆了灵菌红素生物合成基因簇，并在大肠杆菌中实现了异源表达；选择4种大肠杆菌常用启动子驱动灵菌红素基因簇的表达，发现rpoS启动子具有较好的效果。【结论】本研究从甘蓝根际分离得到一株产灵菌红素的黏质沙雷氏菌，完成了全基因组测序和分析，从中克隆得到灵菌红素生物合成基因簇，并成功在大肠杆菌中异源表达。

关键词

甘蓝 / 黏质沙雷氏菌 / 灵菌红素 / 基因组分析 / 生物合成基因簇 / 异源表达

Abstract

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[Objective] To select the elite strains among the microorganisms isolated from the rhizosphere of Brassica oleracea var. capitata and evaluate the secondary metabolism potential of the strains, laying a solid foundation for mining the microbial resources in the rhizosphere of this plant. [Methods] The roots of B. oleracea var. capitata were collected from Xiannüshan Town in Wulong District of Chongqing. The conventional methods were then used to isolate microbial strains, and one strain of Serratia marcescens was selected for genome sequencing (PacBio RS Ⅱ and Illumina HiSeq) and bioinformatic analysis. The antiSMASH was used for the detection and comparison of biosynthetic gene clusters (BGCs) encoding secondary metabolites. Red/ET recombination was employed to capture the BGC for prodigiosin. [Results] One prodigiosin-producing strain of S. marcescens was isolated from the rhizosphere of B. oleracea var. capitata. The genome of this strain contained 11 putative BGCs (1–11) for secondary metabolites, of which 9 BGCs displayed low similarities to the BGCs encoding known compounds. This result suggested that the strain had great potential of producing novel secondary metabolites. Prodigiosin was identified by HPLC and high-resolution mass spectrometry (HRMS). Heterologous expression of BGC4 resulted in the production of prodigiosin in Escherichia coli. Four common promoters were used to drive the expression of BGC in E. coli, and the highest prodigiosin production was observed with the rpoS promoter. [Conclusion] A prodigiosin-producing strain of S. marcescens was isolated from the rhizosphere of B. oleracea var. capitata. The BGC for prodigiosin was captured from the chromosome of this strain and expressed successfully in the surrogate host E. coli BAP1.

Key words

Brassica oleracea var. capitata / Serratia marcescens / prodigiosin / genomic analysis / biosynthetic gene cluster / heterologous expression

引用本文

张雨欣, 王美燕, 李殿怡, 司军, 卞小莹, 牛国清, 刘蓉. 一株产灵菌红素黏质沙雷氏菌的分离鉴定及其基因组分析. 微生物学报, 2024 , 64 (11) : 4290 -4307 . DOI: 10.13343/j.cnki.wsxb.20240303

Yuxin ZHANG, Meiyan WANG, Dianyi LI, Jun SI, Xiaoying BIAN, Guoqing NIU, Rong LIU. Isolation, identification, and genomic analysis of a prodigiosin-producing strain of Serratia marcescens[J]. Acta Microbiologica Sinica, 2024 , 64 (11) : 4290 -4307 . DOI: 10.13343/j.cnki.wsxb.20240303

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结球甘蓝(Brassica oleracea var. capitata)，简称甘蓝，是一种重要的十字花科蔬菜，因其叶球中含有丰富的营养物质而在世界各地普遍种植。据统计，甘蓝在重庆市武隆区、涪陵区和奉节县等高山地区(海拔800−1 800 m)栽培面积达6 500 hm^2[1]，这些区域具有昼夜温差大、土层深厚疏松且富含有机质等独特地理要素，不仅有利于甘蓝种植，而且还塑造了高山地区特有的微生物群落。植物根际微生物在植物生长、营养吸收、胁迫耐受和病原抵御等方面都具有一定的作用，比如枯草芽孢杆菌和荧光假单胞菌促进红甘蓝对微量元素的吸收^[2]，分离自不同作物的沙雷氏菌可以有效地抑制根腐病和黄萎病等植物病害^[3-4]。此外，根际微生物也是活性次级代谢物的重要来源，例如绝大多数从根际分离的微生物产生的非那嗪类化合物对真菌病原体有较好抑制效果^[5]；分离自水稻根际的铜绿假单胞菌BRp3产生2种新的次级代谢产物，对引发叶枯病的病原菌有拮抗作用^[6]；从沙漠植物根际分离的微生物产生的不同结构次级代谢物，对癌细胞均有较强毒性^[7]。

灵菌红素(prodigiosin)是一类含有三吡咯环骨架结构的天然色素，具有抗菌、抗疟疾、抗癌和免疫抑制等生物活性^[8-9]。值得一提的是，灵菌红素对人的肝癌及结肠癌细胞等具有靶向性，而对正常细胞毒性低，是一种具有广泛应用前景的抗癌药物^[10]。同时，灵菌红素能抑制藻类生长，可用于治理海洋赤潮及淡水水华等环境问题^[11]。此外，灵菌红素作为生态染料具有良好着色度和上染率^[12-13]，在纺织业也具有广阔的应用前景。

微生物是灵菌红素的主要来源，其产生菌包括沙雷氏菌属(Serratia)、链霉菌属(Streptomyces)、假单胞菌属(Pseudomonas)和河氏菌属(Hahella)等^[14]。黏质沙雷氏菌是灵菌红素的主要产生菌，该菌是一种革兰氏阴性细菌，在自然界中广泛分布，水和土壤都是该菌的天然生境。黏质沙雷氏菌在临床上被列为人兽共患的机会致病菌^[15]，会侵染受损或低免疫功能机体，导致肺炎、尿路感染和菌血症等^[16-18]，并且对多种常用抗菌药具有一定的耐药性。

灵菌红素通常由pigA−N共14个基因编码的蛋白合成，这些基因组成一个转录单元，并由pigA启动子驱动它们的转录。灵菌红素生物合成途径包括2-甲基-3-戊基吡咯(2-methyl-3-n-amyl-pyrrole, MAP)和4-甲氧基-2, 2-二吡咯-5-羧甲基乙醛(4-methoxy-2, 2′-bipyrrole-5-carbaldehyde, MBC)两个分支途径(图1)。MAP以2-辛烯醛(2-octenal)和丙酮酸(pyruvate)为底物，而MBC以脯氨酸(proline)和丙二酰辅酶A (malonyl-CoA)为底物，两个分支途径最后经缩合酶PigC作用生成灵菌红素^[19]。此外，黏质沙雷氏菌的灵菌红素合成受多种因子的调控^[20]，如LuxI/LuxR和LuxS群体感应系统^[21]，RssA/RssB双组分调控系统^[22]，PsrA、OmpR和RcsB转录调控因子等^[23-24]。

本研究从甘蓝根际分离得到一株黏质沙雷氏菌，全基因组测序和生物信息学分析发现该菌含有一个灵菌红素生物合成基因簇，利用Red/ET重组工程技术克隆了完整的灵菌红素基因簇，并在大肠杆菌中进行异源表达。本研究不仅有助于灵菌红素的工程改造，而且为黏质沙雷氏菌的基因组挖掘奠定了基础。

1 材料与方法

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1.1 材料

1.1.1 样品采集

用于菌株分离的样品采集于重庆市武隆区仙女山镇仙女山村，田间随机采取甘蓝根，装入无菌样品袋中，取回后保存于冰箱备用。

1.1.2 培养基

为了分离甘蓝根际微生物，选择了10种常用微生物培养基^[25]。本研究所用沙雷氏菌培养基包括LB (g/L)：胰蛋白胨10.0，酵母提取物5.0，NaCl 10.0，琼脂15.0；TSB (g/L)：TSB 30.0，琼脂15.0；LBG (g/L)：LB培养基中加入甘油20.0；GTY (g/L)：胰蛋白胨10.0，甘油20.0，琼脂15.0；GYB (g/L)：胰蛋白胨5.0，酵母提取物0.5，葡萄糖10.0，牛肉膏3.0，琼脂15.0；TB (g/L)：胰蛋白胨12.0，酵母提取物24.0，K₂HPO₄ 12.5，KH₂PO₄ 2.3，琼脂15.0。以上培养基均调节pH值至7.0。

1.1.3 菌株

本研究所用菌株见表1。

1.1.4 质粒

本研究所用质粒见表2。

1.1.5 引物

本研究所用引物由生工生物工程(上海)股份有限公司合成，引物序列见表3。

1.1.6 主要试剂和仪器

色谱级甲醇，北京迪科马科技有限公司；氯霉素、卡那霉素，生工生物工程(上海)股份有限公司；KOD Plus Neo，TOYOBO公司；限制性内切酶、T4 DNA连接酶，TaKaRa公司；T4 DNA聚合酶，纽英伦生物技术(北京)有限公司。

电穿孔系统、PCR仪，Bio-Rad公司；恒温培养箱，上海博迅医疗生物仪器股份有限公司；振荡培养箱，上海知楚仪器有限公司；光学显微镜，南京江南永新光学有限公司；高效液相色谱仪，岛津公司；高效液相色谱-高分辨质谱联用系统，安捷伦科技(中国)有限公司。

1.2 菌株分离和鉴定

取出保存的甘蓝根，用灭菌的去离子水冲洗表面泥土，将根分段剪碎，随后取20 g根和100 mL无菌水放入破壁机中破碎至糊状，经4层无菌纱布过滤后的滤液用生理盐水进行逐级稀释。取各梯度稀释液100 μL分别涂布于分离培养基，置于28 ℃培养^[25]。利用通用引物27F/1492R对细菌的16S rRNA基因进行PCR扩增，所得产物经琼脂糖凝胶电泳检测并送至生工生物工程(上海)股份有限公司测序。用BioEdit拼接测序所得基因序列后在NCBI中进行比对。

1.3 黏质沙雷氏菌的培养

挑取黏质沙雷氏菌单菌落，接种至LB液体培养基中，28 ℃、220 r/min培养2 h。待其OD₆₀₀约为0.5时，接种环取菌液在LB、LBG、TB、TSB、GTY和GYB共6种固体培养基上划线，置于28 ℃培养箱培养2 d后观察菌落形态。同时，菌液以2%的接种量接至30 mL上述6种液体培养基中，28 ℃、220 r/min培养6 h后显微观察细菌形态。

1.4 黏质沙雷氏菌的系统发育树构建

利用NCBI对黏质沙雷氏菌的16S rRNA基因序列进行比对，选取沙雷氏菌属中具有较高相似性的序列，通过MEGA 6.0构建系统发育树。

1.5 基因组测序和生物信息学分析

黏质沙雷氏菌的基因组测序由上海美吉生物医药科技有限公司完成，依托美吉云平台(www.i-sanger.com)完成基因组的特征分析。利用antiSMASH 7.0 (http://antismash.secondarymetabolites.org/)对该菌株的次级代谢产物生物合成基因簇进行分析。

1.6 菌株发酵产物分析鉴定

黏质沙雷氏菌接种至LB、LBG、TB、TSB、GTY和GYB共6种培养基，大肠杆菌重组菌株接种至LBG培养基。28 ℃培养2 d后，收集板上菌落，用酸性甲醇(按4%体积比加入1 mol/LHCl)破胞萃取，12 000 r/min离心10 min取上清液，过0.22 μm滤膜后上机测试。HPLC和HPLC-HRMS的流动相A为含0.1%甲酸的去离子水，流动相B为甲醇，检测波长为535 nm。HPLC洗脱程序：0−2 min，50%−100%甲醇；2−5 min，100%甲醇；5−7 min，100%−50%甲醇；7−20 min，50%甲醇，流速1.0 mL/min；HPLC-HRMS洗脱程序：0−3 min，70%甲醇；3−8 min，70%−100%甲醇；8−13 min，100%甲醇；13−15 min，70%甲醇，流速0.3 mL/min。

1.7 灵菌红素基因簇的克隆及异源表达

黏质沙雷氏菌接种至50 mL LB培养基中，恒温摇床中28 ℃、220 r/min条件下培养2 d，收集菌体并提取基因组。利用Nde Ⅰ酶切，获得包含完整基因簇的DNA片段。利用引物对pig-HA-p15A-1-F/R和pig-HA-p15A-2-F/R，以质粒p15A-cm-TetR-TetO为模板，依次进行PCR扩增，最终获得带有80 bp同源臂的约2.7 kb DNA片段。将其与上述基因组酶切回收后的DNA片段混合，电转化至大肠杆菌GB05-dir中^[27]。采用酶切验证确定获得含目标基因簇的重组质粒，并将其命名为p15A: : pig。将p15A: : pig分别转入大肠杆菌DH5α、JM109、BL21(DE3)和BAP1中进行异源表达。

1.8 灵菌红素基因簇的启动子改造

pET28a质粒分别经Nco Ⅰ/BamH Ⅰ和Bgl Ⅱ/BamH Ⅰ酶切制备两种线性化载体。以BW25113基因组为模板，用引物对oxb20-F/R扩增oxb20启动子片段；以大肠杆菌K-12基因组为模板，分别用引物对rpoD-F/R和rpoS-F/R制备rpoD和rpoS启动子片段。以黏质沙雷氏菌BoR121基因组为模板，分别采用引物对pig Up-F1/R、pig Up-F2/R、pig Up-F3/R进行扩增，得到3个500 bp的pigA1、pigA2和pigA3片段作为上游同源臂，用引物对pig Dn-F/R扩增得到530 bp的copA下游同源臂片段。oxb20、rpoD分别与pigA3连接获得oxb20-pigA3和rpoD-pigA3片段。然后，将pigA1、oxb20-pigA3和rpoD-pigA3分别与下游copA片段酶切连接至pET28a线性化载体，得到重组质粒pET28a: : T7 pigUpDn、pET28a: : P_oxb20 pigUpDn和pET28a: : P_rpoD pigUpDn。rpoS与pigA2片段磷酸化连接至pET28a: : P_rpoD pigUpDn (Bgl Ⅱ+Nhe Ⅰ酶切)质粒相应位置，得到重组质粒pET28a: : P_rpoS pigUpDn。将上述构建的4个重组质粒经Nhe Ⅰ线性化，利用PCR-targeting技术将灵菌红素基因簇插入到该线性化载体上，得到重组质粒pET28a: : T7 pig、pET28a: : P_oxb20 pig、pET28a: : P_rpoD pig和pET28a: : P_rpoS pig，之后转入大肠杆菌BAP1。

2 结果与分析

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2.1 黏质沙雷氏菌的分离和鉴定

随机采集了重庆市武隆区仙女山镇仙女山村的甘蓝根，使用传统细菌培养方法对甘蓝根际微生物进行分离和纯培养，在LB平板上观察到一株呈橙色的菌株。使用通用引物27F/1492R对该菌的16S rRNA基因进行PCR扩增，将所得16S rRNA基因序列进行BLAST比对分析，利用MEGA 6.0构建系统发育树，发现该菌株与黏质沙雷氏菌ATCC 274的亲缘关系最近(图2)，其相似性为99.67%，因此将其命名为黏质沙雷氏菌BoR121。为了进一步观察其菌落形态，将该菌分别接种在6种不同的固体培养基上(LB、LBG、TSB、GTY、GYB和TB)，28 ℃培养2 d后菌落表面光滑、湿润、黏稠，呈圆形凸起，该菌在6种培养基上呈现不同的颜色(图3A)。此外，我们将该菌株分别接种至上述对应的液体培养基中，28 ℃培养6 h后进行显微观察，发现该菌在6种液体培养基中均呈短杆状(图3B)。以上结果表明，黏质沙雷氏菌BoR121是一株短杆状细菌，具有产生有颜色化合物的能力。

2.2 基因组测序及分析

为了深入了解黏质沙雷氏菌BoR121的生物学特性，对其基因组进行了测序。Illumina HiSeq二代测序质控数据显示，原始reads读长150 bp，质控后得到高质量reads用来进行基因组组装，Circos基因组如图4所示。PacBio三代测序结果显示，总reads的数量为136 179，平均reads读长8 025.33 bp，总碱基长1 092 881 030 bp。完整基因组信息表明，该菌株为黏质沙雷氏菌，其基因组为一条环状染色体，全长5 228 182 bp，G+C含量为59.51%。预测有4 996个编码基因，平均长度913.94 bp，总长度为4 566 027 bp，占基因组总长度的87.33%。基因组包括22种tRNA基因共92个；22个rRNA基因中，16S rRNA基因和23S rRNA基因各7个(表4)。将预测得到的基因集与各种功能数据库进行比对，分析发现KEGG数据库中共注释到3 098个基因，占编码基因数目的62.01%，根据其功能分为6大类，分别是胞内生命过程(cellular processes)、环境信息处理(environmental information processing)、遗传信息处理(genetic information processing)、人类疾病(human diseases)、代谢(metabolism)和有机体生命系统(organismal systems)，其中代谢类相关基因有1 506个(图5)。COG数据库中共注释到4 502个基因，占编码基因数目的90.11%，共有21种类型，除了未知功能基因外，氨基酸转运和代谢类(amino acid transport and metabolism)以及转录类(transcription)基因数目最多，分别为427个和414个(图6)。GO数据库中共注释到3 136个基因，占编码基因的62.77%，根据功能的不同，这些基因被分为生物过程(biological process)，细胞成分(cellular component)和分子功能(molecular function)三大类(图7)。

为了评估黏质沙雷氏菌BoR121合成次级代谢产物的能力，本研究通过antiSMASH分析其基因组序列，发现11个次级代谢产物生物合成基因簇，依次命名为基因簇1−11 (表5)，其中3个属于非核糖体肽合成酶类(non-ribosomal peptide synthetase, NRPS)、2个非核糖体肽合成酶-聚酮合酶杂合类(non-ribosomal peptide synthetase-polyketide synthetase, NRPS-PKS)、2个硫肽类(thiopeptide)、2个内酯类(lactone)和2个其他类型。其中，基因簇4与黏质沙雷氏菌ATCC 274的灵菌红素基因簇有100%的相似性，与沙雷氏菌ATCC 39006的灵菌红素基因簇有82%的相似性^[29]，基因簇6与来自Yersinia pestis CO92的Yersinopine基因簇有100%的相似性^[30]，其他9个基因簇与已报道化合物编码基因簇相似性较低，说明该菌具有产生多种新结构、新功能化合物的潜力。

2.3 灵菌红素的鉴定

结合黏质沙雷氏菌BoR121的形态观察及基因组序列分析，确认该菌具有合成灵菌红素的能力。从平板上刮取菌落，使用酸性甲醇破胞萃取，将所得萃取液进行HPLC分析，结果显示，6种培养基上收集的样品均在535 nm吸收波长处观察到明显的单一峰，该峰的保留时间为8.4 min，与灵菌红素标准样品一致(图8A)。HRMS测定其分子量为324.235 6 [M+H]⁺，与灵菌红素理论分子量(324.199 8 [M+H]⁺)相符，确定该化合物为灵菌红素(图8B)。

2.4 灵菌红素基因簇的克隆

黏质沙雷氏菌BoR121基因组中含有灵菌红素合成基因簇(基因簇4)，该基因簇由14个基因组成一个转录单元，其基因编码产物对应PigA−N的同源蛋白(表6)。选择其他沙雷氏菌(S. marcescens ATCC 274、S. plymuthica strain AS13和Serratia sp. ATCC 39006)和河氏菌(H. chejuensis KCTC 2396)中灵菌红素基因簇进行比较分析，发现该基因簇的排布方式与S. marcescens ATCC 274一致(图9)。为进一步确认该基因簇负责灵菌红素的生物合成，通过Red/ET重组工程技术直接从该菌染色体上钓取了大小约为26.3 kb的DNA片段，其中包含了大小为20.9 kb的灵菌红素基因簇，得到重组质粒p15A: : pig (图10A)。该质粒分别经EcoR Ⅰ和Sac Ⅰ酶切后，琼脂糖凝胶电泳检测发现酶切条带与预期相符(图10B)。结果表明，本研究成功克隆得到了灵菌红素基因簇。

2.5 灵菌红素基因簇的异源表达

将重组质粒p15A: : pig分别导入大肠杆菌DH5α、JM109、BL21(DE3)和BAP1中，得到相应的重组菌株DH5α-pig、JM109-pig、BL21-pig和BAP1-pig。将这些重组菌株分别接种到LBG平板上，表型观察发现仅BAP1-pig呈现明显的红色(图11A)，表明灵菌红素基因簇在大肠杆菌BAP1中成功异源表达。本研究选择了4个大肠杆菌常用启动子T7、P_oxb20、P_rpoD和P_rpoS，用于替换基因簇的原始启动子(图11B)，获得了BAP1-T7-pig、BAP1-oxb20-pig、BAP1-rpoD-pig和BAP1-rpoS-pig重组菌株。所得菌株分别在LBG培养基上进行固体发酵后，刮取菌体经酸性甲醇破胞萃取，萃取液呈现不同程度红色。HPLC分析结果显示，除BAP1-T7-pig外，其余3株菌均检测到灵菌红素，其中BAP1-rpoS-pig产量最高，约101.0 μg/g (图11C、11D)。

3 讨论与结论

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来源于根际的优异菌株不仅可以被加工成微生物肥料及微生物农药等生物制剂^[31]，还是挖掘许多新型次级代谢产物的资源库。本研究从甘蓝根分离得到一株产红色素的微生物，通过基因组测序和生物信息学分析发现该菌为黏质沙雷氏菌。该菌株共有11个次级代谢产物生物合成基因簇，其中2个与已知化合物灵菌红素和Yersinopine合成基因簇相似性高，另外9个与现有化合物编码基因簇相似性较低，表明该菌还具有合成新化合物的潜力，本研究为开发新功能性化合物提供了新菌种资源。

灵菌红素及其衍生物具有多种医药活性和广泛应用前景^[32-33]，因而国内外研究者一直致力于实现这些化合物的绿色高效合成，但是其天然宿主黏质沙雷氏菌是机会致病菌且遗传背景不清晰，难以进行基因操作，因此现有研究主要集中在基因簇的异源表达^[34-35]。目前具有遗传背景清晰、遗传操作简单、生物安全的常用异源宿主包括大肠杆菌、链霉菌和酿酒酵母^[36-37]，本研究选择了大肠杆菌作为异源表达宿主。本研究通过Red/ET重组工程技术，克隆得到了完整的灵菌红素基因簇，该基因簇在大肠杆菌BAP1中表达，推测是因为BAP1基因组中特有的sfp基因，该基因编码的磷酸泛酰巯基乙胺基转移酶(PPTase)对PKS和NRPS类化合物酰基化起到了关键作用^[38]。以上分析表明选择含有PPTase的宿主，有利于PKS和NRPS类化合物异源表达。后续在大肠杆菌BAP1中引入修饰基因或采用组合生物合成等手段，有望拓展灵菌红素的结构多样性。

优化调控元件、增加基因簇拷贝数和启动子改造是提高外源基因表达效率^[39-41]，高效合成天然产物的通用策略。鉴于灵菌红素基因簇中基因形成一个转录单元，本研究将灵菌红素基因簇pigA启动子替换为大肠杆菌中的常用启动子，发现不同启动子表达产量存在差异，其中BAP1-oxb20-pig和BAP1-rpoS-pig重组菌株的产量较高，推测大肠杆菌内源启动子有利于该基因簇的高效表达。为进一步提高灵菌红素产量，挖掘异源宿主内源强启动子是一种有效途径。相比黏质沙雷氏菌，大肠杆菌中灵菌红素的产量仍偏低，推测是由于内源供给生物合成前体不足。采用代谢工程改造及内源前体物的生物传感器动态调控系统，提高胞内关键中间体水平，有望实现目标化合物的增产。

基金

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国家重点研发计划(2020YFA0907700)

参考文献

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文献

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参考文献引证文献

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2024年第64卷第11期

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文章信息

doi: 10.13343/j.cnki.wsxb.20240303

接收时间：2024-05-15
首发时间：2026-03-21
出版时间：2024-07-06

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收稿日期：2024-05-15
录用日期：2024-07-03

基金

National Key Research and Development Program of China(2020YFA0907700)

国家重点研发计划(2020YFA0907700)

作者信息

1 西南大学农学与生物科技学院, 重庆 400715

2 重庆中烟工业有限责任公司, 烟草资源科学利用重庆重点实验室, 重庆 400060

3 西南大学园艺园林学院, 长江上游农业生物安全与绿色生产教育部重点实验室, 重庆 400715

4 山东大学, 微生物技术国家重点实验室, 山东青岛 266237

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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Strains	Genotype/Description	Reference/Source
Serratia marcescens BoR121	Wild-type, prodigiosin producer	This work
Escherichia coli
DH5α	F^–, φ80lacZΔM15, Δ(lacZYA-argF) U169, recA1, endA1, hsdR17 (rK^–, mK⁺), phoA, supE44, λ^–, thi-1, gyrA96, relA1	Thermo Fisher Scientific
JM109	endA1, recA1, gyrA96, thi, hsdR17 (r_k^–, m_k⁺), relA1, supE44, Δ(lac-proAB), [F′ traD36, proAB, lacI^qZΔM15]	Thermo Fisher Scientific
BL21(DE3)	fhuA2, ompT, gal (λ DE3), [dcm] ∆hsdS, λ DE3=λ sBamH Io ∆EcoR Ⅰ-B int-(lacI-PlacUV5-T7 gene1) i21 ∆nin5	New England Biolabs
BAP1	BL21(DE3), ΔprpRBCD-T7prom-sfp, T7prom-prpE	Kerafast
BW25113/pIJ790	K-12 derivative, ΔaraBAD, ΔrhaBAD/pIJ790	[26]
GB05-dir	Red/ET mediated linear-linear recombineering	[27-28]
DH5α-pig	E. coli DH5α containing p15A: : pig	This work
JM109-pig	E. coli JM109 containing p15A: : pig	This work
BL21-pig	E. coli BL21(DE3) containing p15A: : pig	This work
BAP1-pig	E. coli BAP1 containing p15A: : pig	This work
BAP1-T7-pig	E. coli BAP1 containing pET28a: : T7 pig	This work
BAP1-rpoD-pig	E. coli BAP1 containing pET28a: : P_rpoD pig	This work
BAP1-rpoS-pig	E. coli BAP1 containing pET28a: : P_rpoS pig	This work
BAP1-oxb20-pig	E. coli BAP1 containing pET28a: : P_oxb20 pig	This work

Strains

Genotype/Description

Reference/Source

Serratia marcescens BoR121

Wild-type, prodigiosin producer

This work

Escherichia coli

DH5α

F^–, φ80lacZΔM15, Δ(lacZYA-argF) U169, recA1, endA1, hsdR17 (rK^–, mK⁺), phoA, supE44, λ^–, thi-1, gyrA96, relA1

Thermo Fisher Scientific

JM109

endA1, recA1, gyrA96, thi, hsdR17 (r_k^–, m_k⁺), relA1, supE44, Δ(lac-proAB), [F′ traD36, proAB, lacI^qZΔM15]

Thermo Fisher Scientific

BL21(DE3)

fhuA2, ompT, gal (λ DE3), [dcm] ∆hsdS, λ DE3=λ sBamH Io ∆EcoR Ⅰ-B int-(lacI-PlacUV5-T7 gene1) i21 ∆nin5

New England Biolabs

BAP1

BL21(DE3), ΔprpRBCD-T7prom-sfp, T7prom-prpE

Kerafast

BW25113/pIJ790

K-12 derivative, ΔaraBAD, ΔrhaBAD/pIJ790

[26]

GB05-dir

Red/ET mediated linear-linear recombineering

[27-28]

DH5α-pig

E. coli DH5α containing p15A: : pig

This work

JM109-pig

E. coli JM109 containing p15A: : pig

This work

BL21-pig

E. coli BL21(DE3) containing p15A: : pig

This work

BAP1-pig

E. coli BAP1 containing p15A: : pig

This work

BAP1-T7-pig

E. coli BAP1 containing pET28a: : T7 pig

This work

BAP1-rpoD-pig

E. coli BAP1 containing pET28a: : P_rpoD pig

This work

BAP1-rpoS-pig

E. coli BAP1 containing pET28a: : P_rpoS pig

This work

BAP1-oxb20-pig

E. coli BAP1 containing pET28a: : P_oxb20 pig

This work

Plasmids	Relevant characteristics	Reference/Source
p15A-cm-TetR-TetO	ori (p15A), Cm^R	[27]
pET28a	ori (pBR322), T7, His6·tag, Kan^R	Novagen
p15A: : pig	A derivative of p15A containing the prodigiosin biosynthetic gene cluster, Cm^R	This work
pET28a: : T7 pigUpDn	pET28a containing T7 promoter, 500 bp of pigA1 and 530 bp of copA	This work
pET28a: : P_rpoD pigUpDn	pET28a containing P_rpoD, 500 bp of pigA3 and 530 bp of copA	This work
pET28a: : P_oxb20 pigUpDn	pET28a containing P_oxb20, 500 bp of pigA3 and 530 bp of copA	This work
pET28a: : P_rpoS pigUpDn	pET28a containing P_rpoS, 500 bp of pigA2 and 530 bp of copA	This work
pET28a: : T7 pig	A derivative of pET28a containing the prodigiosin biosynthetic gene cluster driven by T7 promoter	This work
pET28a: : P_oxb20 pig	A derivative of pET28a containing the prodigiosin biosynthetic gene cluster driven by oxb20 promoter	This work
pET28a: : P_rpoD pig	A derivative of pET28a containing the prodigiosin biosynthetic gene cluster driven by rpoD promoter	This work
pET28a: : P_rpoS pig	A derivative of pET28a containing the prodigiosin biosynthetic gene cluster driven by rpoS promoter	This work

Plasmids

Relevant characteristics

Reference/Source

p15A-cm-TetR-TetO

ori (p15A), Cm^R

[27]

pET28a

ori (pBR322), T7, His6·tag, Kan^R

Novagen

p15A: : pig

A derivative of p15A containing the prodigiosin biosynthetic gene cluster, Cm^R

This work

pET28a: : T7 pigUpDn

pET28a containing T7 promoter, 500 bp of pigA1 and 530 bp of copA

This work

pET28a: : P_rpoD pigUpDn

pET28a containing P_rpoD, 500 bp of pigA3 and 530 bp of copA

This work

pET28a: : P_oxb20 pigUpDn

pET28a containing P_oxb20, 500 bp of pigA3 and 530 bp of copA

This work

pET28a: : P_rpoS pigUpDn

pET28a containing P_rpoS, 500 bp of pigA2 and 530 bp of copA

This work

pET28a: : T7 pig

A derivative of pET28a containing the prodigiosin biosynthetic gene cluster driven by T7 promoter

This work

pET28a: : P_oxb20 pig

A derivative of pET28a containing the prodigiosin biosynthetic gene cluster driven by oxb20 promoter

This work

pET28a: : P_rpoD pig

A derivative of pET28a containing the prodigiosin biosynthetic gene cluster driven by rpoD promoter

This work

pET28a: : P_rpoS pig

A derivative of pET28a containing the prodigiosin biosynthetic gene cluster driven by rpoS promoter

This work

Primers	Sequences (5′→3′)	Purpose
Underlined sequences for restriction enzyme recognition sites, and lowercase letters for overlapping between DNA sequences.
27F	AGAGTTTGATCCTGGCTCAG	PCR amplification of 16S rRNA gene
1492R	GGTTACCTTGTTACGCTT	PCR amplification of 16S rRNA gene
pig-HA-p15A-1-F	gccgggcgagcgtcggctccatctggtgggcagcatccaAGATCCGAAAACCCCAAGTT	p15A vector fragment carrying the 80 bp target gene cluster homology arm
pig-HA-p15A-1-R	atgggaataaaagtaagcatgcaaacctcctgtcagacaAGATCCTTTCTCCTCTTTAG
pig-HA-p15A-2-F	tcgcccaaaagttacgcttatccggcgctggacatcactctGCCGGGCGAGCGTCGGCT
pig-HA-p15A-2-R	tgacgccggcgaacaccacaatcagcgcgacgacgatcacgATGGGAATAAAAGTAAGC
oxb20-F	AATTAGATCTCTATTTACAAGAGGGGGGCG	PCR 706 bp of oxb20
oxb20-R	GCCGGGTAATACCGGATAGT	PCR 706 bp of oxb20
rpoD-F	AATTAGATCTACGCACGACCATGCGTATAC	PCR 600 bp of rpoD
rpoD-R	tgataagttaaaatccatAAGACGGTATCCACACTTAT	PCR 600 bp of rpoD
rpoS-F	AATTAGATCTCTTTAGTGAAGGGTGAGCTGGC	PCR 1 367 bp of rpoS
rpoS-R	AAGGTGGCTCCTACCCGTGA	PCR 1 367 bp of rpoS
pig Up-F1	AATTCCATGGATGGATTTTAACTTATCAAGTGGGC	PCR 500 bp of pigA1
pig Up-F2	ATGGATTTTAACTTATCAAGTGGGC	PCR 500 bp of pigA2
pig Up-F3	tatccggtattacccggcATGGATTTTAACTTATCAAGTGGG	PCR 500 bp of pigA3
pig Up-R	TTAAGCTAGCATGTCGGCAATCGGCGCGTT	PCR 500 bp of pigA1−A3
pig Dn-F	AATTGCTAGCCCCCAACGCGGCAGCGCGGTT	PCR 530 bp of copA
pig Dn-R	AATTGAATTCGGATCCCTTTGCGTTGTCGATCTCG	PCR 530 bp of copA

Primers

Sequences (5′→3′)

Purpose

Underlined sequences for restriction enzyme recognition sites, and lowercase letters for overlapping between DNA sequences.

27F

AGAGTTTGATCCTGGCTCAG

PCR amplification of 16S rRNA gene

1492R

GGTTACCTTGTTACGCTT

PCR amplification of 16S rRNA gene

pig-HA-p15A-1-F

gccgggcgagcgtcggctccatctggtgggcagcatccaAGATCCGAAAACCCCAAGTT

p15A vector fragment carrying the 80 bp target gene cluster homology arm

pig-HA-p15A-1-R

atgggaataaaagtaagcatgcaaacctcctgtcagacaAGATCCTTTCTCCTCTTTAG

pig-HA-p15A-2-F

tcgcccaaaagttacgcttatccggcgctggacatcactctGCCGGGCGAGCGTCGGCT

pig-HA-p15A-2-R

tgacgccggcgaacaccacaatcagcgcgacgacgatcacgATGGGAATAAAAGTAAGC

oxb20-F

AATTAGATCTCTATTTACAAGAGGGGGGCG

PCR 706 bp of oxb20

oxb20-R

GCCGGGTAATACCGGATAGT

PCR 706 bp of oxb20

rpoD-F

AATTAGATCTACGCACGACCATGCGTATAC

PCR 600 bp of rpoD

rpoD-R

tgataagttaaaatccatAAGACGGTATCCACACTTAT

PCR 600 bp of rpoD

rpoS-F

AATTAGATCTCTTTAGTGAAGGGTGAGCTGGC

PCR 1 367 bp of rpoS

rpoS-R

AAGGTGGCTCCTACCCGTGA

PCR 1 367 bp of rpoS

pig Up-F1

AATTCCATGGATGGATTTTAACTTATCAAGTGGGC

PCR 500 bp of pigA1

pig Up-F2

ATGGATTTTAACTTATCAAGTGGGC

PCR 500 bp of pigA2

pig Up-F3

tatccggtattacccggcATGGATTTTAACTTATCAAGTGGG

PCR 500 bp of pigA3

pig Up-R

TTAAGCTAGCATGTCGGCAATCGGCGCGTT

PCR 500 bp of pigA1−A3

pig Dn-F

AATTGCTAGCCCCCAACGCGGCAGCGCGGTT

PCR 530 bp of copA

pig Dn-R

AATTGAATTCGGATCCCTTTGCGTTGTCGATCTCG

PCR 530 bp of copA

Feature	Chromosome characteristics
Chromosome size (bp)	5 228 182
G+C content (%)	59.51
Predicted genes	4 996
rRNA operons	22
tRNA genes	92
sRNA genes	112
Genes assigned to COG	4 502
Genes assigned to GO	3 136
Genes assigned to KEGG	3 098
CRISPR repeat regions	4
Prophages	3
Simple tandem repeats	26
Interspersed repeats	72
Genomic islands	8
Carbohydrate-active enzymes	138
Transposable element	9
Antibiotic resistance genes	322
Virulence factors	656
Secondary metabolite gene clusters	11

Feature

Chromosome characteristics

Chromosome size (bp)

5 228 182

G+C content (%)

59.51

Predicted genes

4 996

rRNA operons

tRNA genes

sRNA genes

112

Genes assigned to COG

4 502

Genes assigned to GO

3 136

Genes assigned to KEGG

3 098

CRISPR repeat regions

Prophages

Simple tandem repeats

Interspersed repeats

Genomic islands

Carbohydrate-active enzymes

138

Transposable element

Antibiotic resistance genes

322

Virulence factors

656

Secondary metabolite gene clusters

Cluster	Type	From	To	Most similar known biosynthetic gene cluster
1	Hserlactone	23 358	44 032	Unknown
2	NRPS	278 246	342 472	Unknown
3	Betalactone	747 506	773 174	Unknown
4	NRPS-PKS	1 174 609	1 195 556	Prodigiosin
5	Thiopeptide	1 797 266	1 823 709	Unknown
6	Other	2 183 729	2 205 826	Yersinopine
7	Other	2 467 098	2 487 376	Unknown
8	NRPS-PKS	2 514 439	2 584 366	Unknown
9	NRPS	3 218 021	3 294 757	Unknown
10	Thiopeptide	3 840 325	3 887 793	Unknown
11	NRPS	4 687 524	4 731 463	Unknown

Cluster

Type

From

Most similar known biosynthetic gene cluster

Hserlactone

23 358

44 032

Unknown

NRPS

278 246

342 472

Unknown

Betalactone

747 506

773 174

Unknown

NRPS-PKS

1 174 609

1 195 556

Prodigiosin

Thiopeptide

1 797 266

1 823 709

Unknown

Other

2 183 729

2 205 826

Yersinopine

Other

2 467 098

2 487 376

Unknown

NRPS-PKS

2 514 439

2 584 366

Unknown

NRPS

3 218 021

3 294 757

Unknown

Thiopeptide

3 840 325

3 887 793

Unknown

NRPS

4 687 524

4 731 463

Unknown

ORF	Number of amino acids	Proposed function	Protein homology	Amino acid identity (%)
pigA	385	l-prolyl-PCP dehydrogenase	Serratia marcescens acyl-CoA dehydrogenase family protein (MDV2100735.1)	383/385 (99)
pigB	670	Oxidoreductase	Serratia marcescens FAD-dependent oxidoreductase (HEJ0404376.1)	667/670 (99)
pigC	888	Prodigiosin synthesizing transferase	Serratia marcescens phosphoenolpyruvate synthase (MDR2288025.1)	884/888 (99)
pigD	867	Thiamine diphosphate dependent-3-acetyloctanal synthase	Serratia marcescens biosynthesis protein PigD (WP_197787955.1)	864/867 (99)
pigE	853	Aminotransferase	Serratia sp. JKS296 minotransferase class Ⅲ-fold pyridoxal phosphate-dependent enzyme (WP_097102633.1)	851/853 (99)
pigF	338	S-adenosyl-l-methionine-dependent methyl transferase	Serratia marcescens methyltransferase domain-containing protein (MBH2772015.1)	336/338 (99)
pigG	87	Acyl carrier protein	Serratia marcescens acyl carrier protein (WP_176063587.1)	86/87 (99)
pigH	648	4-hydroxy-2, 2′-bipyrrole-5-methanol synthase	Serratia sp. JKS296 aminotransferase class Ⅰ/Ⅱ-fold pyridoxal phosphate-dependent enzyme (WP_097102635.1)	643/648 (99)
pigI	491	l-proline-[l-prolyl-carrier protein] ligase	Serratia marcescens d-alanine-poly(phosphoribitol) ligase (AWO78120.1)	489/491 (99)
pigJ	762	Beta-ketoacyl synthase	Serratia marcescens polyketide synthase (WP_261420020.1)	758/762 (99)
pigK	104	Sterol 3-beta-glucosyltransferase	Serratia marcescens RedY (WP_164052918.1)	103/104 (99)
pigL	215	4′-phosphopantetheinyl transferase Sfp	Serratia marcescens 4′-phosphopantetheinyl transferase superfamily protein (WP_306376640.1)	212/215 (99)
pigM	352	Probable NAD(P)H nitroreductase	Serratia marcescens prodigiosin biosynthesis protein PigM (WP_229597835.1)	350/352 (99)
pigN	242	Delta (14)-sterol reductase	Serratia marcescens putative oxidoreductase (QWV59760.1)	240/242 (99)

ORF

Number of amino acids

Proposed function

Protein homology

Amino acid identity (%)

pigA

385

l-prolyl-PCP dehydrogenase

Serratia marcescens acyl-CoA dehydrogenase family protein (MDV2100735.1)

383/385 (99)

pigB

670

Oxidoreductase

Serratia marcescens FAD-dependent oxidoreductase (HEJ0404376.1)

667/670 (99)

pigC

888

Prodigiosin synthesizing transferase

Serratia marcescens phosphoenolpyruvate synthase (MDR2288025.1)

884/888 (99)

pigD

867

Thiamine diphosphate dependent-3-acetyloctanal synthase

Serratia marcescens biosynthesis protein PigD (WP_197787955.1)

864/867 (99)

pigE

853

Aminotransferase

Serratia sp. JKS296 minotransferase class Ⅲ-fold pyridoxal phosphate-dependent enzyme (WP_097102633.1)

851/853 (99)

pigF

338

S-adenosyl-l-methionine-dependent methyl transferase

Serratia marcescens methyltransferase domain-containing protein (MBH2772015.1)

336/338 (99)

pigG

Acyl carrier protein

Serratia marcescens acyl carrier protein (WP_176063587.1)

86/87 (99)

pigH

648

4-hydroxy-2, 2′-bipyrrole-5-methanol synthase

Serratia sp. JKS296 aminotransferase class Ⅰ/Ⅱ-fold pyridoxal phosphate-dependent enzyme (WP_097102635.1)

643/648 (99)

pigI

491

l-proline-[l-prolyl-carrier protein] ligase

Serratia marcescens d-alanine-poly(phosphoribitol) ligase (AWO78120.1)

489/491 (99)

pigJ

762

Beta-ketoacyl synthase

Serratia marcescens polyketide synthase (WP_261420020.1)

758/762 (99)

pigK

104

Sterol 3-beta-glucosyltransferase

Serratia marcescens RedY (WP_164052918.1)

103/104 (99)

pigL

215

4′-phosphopantetheinyl transferase Sfp

Serratia marcescens 4′-phosphopantetheinyl transferase superfamily protein (WP_306376640.1)

212/215 (99)

pigM

352

Probable NAD(P)H nitroreductase

Serratia marcescens prodigiosin biosynthesis protein PigM (WP_229597835.1)

350/352 (99)

pigN

242

Delta (14)-sterol reductase

Serratia marcescens putative oxidoreductase (QWV59760.1)

240/242 (99)