微生物学报

Genes name	Primer sequences (5′→3′)
Amino-acid-N-acetyltransferase (argA)	F: CACGGTGTAGAGCTTAGCGT; R: CAGCATGTTGTGCGACTTCC
N-acetylglutamate kinase (argB)	F: GTGCGGTCATCTCAGGACTT; R: CTTTGTGAACGGCATGCGAG
N-acetyl-γ-glutamyl-phosphate reductase (argC)	F: CTTTGACGCTGCGAGTTTCC; R: CCAGCGAATCGATGAAAGCC
Aspartate aminotransferase (argD)	F: GAAAGTGCGGCCATGGAAAG; R: CACTGAGCGTACTTTTGCCG
N-acetylornithine deacetylase (argE)	F: CGTTGAAATCACCACCGAGC; R: TGAGGTGTTTCTAGCGCCTG
Ornithine transcarbamylase (argF)	F: CGTTAATCACAGGCACGCTG; R: AGAGCCGATTGAAGACACCG
Argininosuccinate synthase (argG)	F: CGCTTAGCAATCAACGGACG; R: AGGTTGTGCTGGCGTATTCA
Argininosuccinate lyase (argH)	F: CACGCTCTTCATCGGTCAGT; R: AGCCAAGCTACGAACCAGTC
Phosphopantothenoylcysteine decarboxylase (coaBC)	F: GGCGTTGGTTAGTAGGCAGT; R: AAGCGGCCCAGTTAATCTCC
Pantetheine-phosphate adenylyltransferase (coaD)	F: CCTATCACCAACGGCCACTT; R: TCAAGACTAGGCTGCTTGCC
Dephospho-CoA kinase (coaE)	F: CCGAACACTTTGGCACAGAC; R: GTGCGTGATAATTGCAGCGA
Citrate synthase (gltA)	F: TGAGCCTGCTAAGCGTACTG; R: GTTCAGCAATCCGTGCGAAG
Malate dehydrogenase (mdh)	F: GTGCTTGCCGGTTTTCTGAG; R: GCGTGTACTCGTTGTCGGTA
Branched-chain amino acid transporter (livH)	F: ATGCCTCCTTACCGAACAGC; R: GGTGCGATTCTTACTGGCCT
Aspartate kinase (lysC)	F: GAGGACGCTATGGAAGAAC; R: GCGATAGGACCAAGAATACG
Aspartate-semialdehyde dehydrogenase (asd)	F: GAACGACAAAGACGCTACAG; R: CAACACTGAAGGCTGACAAG
Diaminobutyrate acetyl transferase (ectA)	F: AGTCGCTGATGCTGTGGTTGG; R: ACATCAAGCGGCGGACAAG
Diaminobutyrate transaminase (ectB)	F: TGGTATTGATGTTGTCTCTGG; R: TCACTACTTCGCCGTCTTGG
Ectoine synthase (ectC)	F: TGAAGGCGAAGGCGAAGTAG; R: GATGTTCGTCGTGCTGATCC
Ectoine hydroxylase (ectD)	F: CCAGAAAGCCACGATATTC; R: TGATGCACGTAAGGATTAGCG
Housekeeping gene (GAPDH)	F: TCCTTCCCTAAACTCGCACCT; R: ACGATAGTAGCGTCAGCAT

Genes name	Primer sequences (5′→3′)
Amino-acid-N-acetyltransferase (argA)	F: CACGGTGTAGAGCTTAGCGT; R: CAGCATGTTGTGCGACTTCC
N-acetylglutamate kinase (argB)	F: GTGCGGTCATCTCAGGACTT; R: CTTTGTGAACGGCATGCGAG
N-acetyl-γ-glutamyl-phosphate reductase (argC)	F: CTTTGACGCTGCGAGTTTCC; R: CCAGCGAATCGATGAAAGCC
Aspartate aminotransferase (argD)	F: GAAAGTGCGGCCATGGAAAG; R: CACTGAGCGTACTTTTGCCG
N-acetylornithine deacetylase (argE)	F: CGTTGAAATCACCACCGAGC; R: TGAGGTGTTTCTAGCGCCTG
Ornithine transcarbamylase (argF)	F: CGTTAATCACAGGCACGCTG; R: AGAGCCGATTGAAGACACCG
Argininosuccinate synthase (argG)	F: CGCTTAGCAATCAACGGACG; R: AGGTTGTGCTGGCGTATTCA
Argininosuccinate lyase (argH)	F: CACGCTCTTCATCGGTCAGT; R: AGCCAAGCTACGAACCAGTC
Phosphopantothenoylcysteine decarboxylase (coaBC)	F: GGCGTTGGTTAGTAGGCAGT; R: AAGCGGCCCAGTTAATCTCC
Pantetheine-phosphate adenylyltransferase (coaD)	F: CCTATCACCAACGGCCACTT; R: TCAAGACTAGGCTGCTTGCC
Dephospho-CoA kinase (coaE)	F: CCGAACACTTTGGCACAGAC; R: GTGCGTGATAATTGCAGCGA
Citrate synthase (gltA)	F: TGAGCCTGCTAAGCGTACTG; R: GTTCAGCAATCCGTGCGAAG
Malate dehydrogenase (mdh)	F: GTGCTTGCCGGTTTTCTGAG; R: GCGTGTACTCGTTGTCGGTA
Branched-chain amino acid transporter (livH)	F: ATGCCTCCTTACCGAACAGC; R: GGTGCGATTCTTACTGGCCT
Aspartate kinase (lysC)	F: GAGGACGCTATGGAAGAAC; R: GCGATAGGACCAAGAATACG
Aspartate-semialdehyde dehydrogenase (asd)	F: GAACGACAAAGACGCTACAG; R: CAACACTGAAGGCTGACAAG
Diaminobutyrate acetyl transferase (ectA)	F: AGTCGCTGATGCTGTGGTTGG; R: ACATCAAGCGGCGGACAAG
Diaminobutyrate transaminase (ectB)	F: TGGTATTGATGTTGTCTCTGG; R: TCACTACTTCGCCGTCTTGG
Ectoine synthase (ectC)	F: TGAAGGCGAAGGCGAAGTAG; R: GATGTTCGTCGTGCTGATCC
Ectoine hydroxylase (ectD)	F: CCAGAAAGCCACGATATTC; R: TGATGCACGTAAGGATTAGCG
Housekeeping gene (GAPDH)	F: TCCTTCCCTAAACTCGCACCT; R: ACGATAGTAGCGTCAGCAT

Analysis content	Wild strain XH26	Mutant strain G₉-72
Molecule shape	Circular	Circular/Oval
Coverage (%)	100	100
Genome size (bp)	4 112 053	4 058 888
G+C content (%)	52.62	52.55
Coding sequences (CDS)	104 708	126 226
Total RNA genes	97	97
tRNAs	63	63
5S rRNAs	6	6
16S rRNAs	6	6
23S rRNAs	6	6
Other RNA genes	16	16
Predicted coding genes	3 927	3 882
Total length of predicted coding genes (bp)	3 687 732	3 636 840

Analysis content	Wild strain XH26	Mutant strain G₉-72
Molecule shape	Circular	Circular/Oval
Coverage (%)	100	100
Genome size (bp)	4 112 053	4 058 888
G+C content (%)	52.62	52.55
Coding sequences (CDS)	104 708	126 226
Total RNA genes	97	97
tRNAs	63	63
5S rRNAs	6	6
16S rRNAs	6	6
23S rRNAs	6	6
Other RNA genes	16	16
Predicted coding genes	3 927	3 882
Total length of predicted coding genes (bp)	3 687 732	3 636 840

Number	ORF number	Protein name	Locus	Block	Reference	Change	Type
− means there are no base pairs at this location.
1	orf00034	RNA-binding protein S1	37 964	Exonic	−	G	Insertion
2	orf00080	Hypothetical protein HALTITAN3299	86 078	Upstream	−	T	Insertion
3	orf00256	Lactoylglutathione lyase	257 737	Upstream	−	C	Insertion
4	orf00263	Hypothetical protein	261 715	Upstream	−	T	Insertion
5	orf00263	Hypothetical protein	261 744	Upstream	−	AG	Insertion
6	orf00469	Acetaldehyde dehydrogenase	471 178	Exonic	T	C	Synonymous SNV
7	orf00501	TRAP transporter substrate-binding protein	504 022	Upstream	−	G	Insertion
8	orf00566	Phosphopantothenoylcysteine decarboxylase	568 137	Exonic	C	A	Nonsynonymous SNV
9	orf00643	ABC transporter ATP-binding protein	647 351	Upstream	A	T	Synonymous SNV
10	orf00726	TRAP transporter large permease	732 078	Exonic	−	GT	Insertion
11	orf01257	UvrY/SirA/GacA family response regulator transcription factor	1 312 231	Upstream	−	A	Insertion
12	orf02141	Branched-chain amino acid ABC transporter permease	2 212 367	Exonic	−	C	Insertion
13	orf02185	Hypothetical protein	2 262 995	Upstream	−	G	Insertion
14	orf02433	Hypothetical protein	2 529 845	Exonic	−	T	Insertion
15	orf02522	Membrane proteins	2 628 488	Exonic	−	C	Insertion
16	orf02541	Ig-like domain-containing protein	2 650 751	Exonic	G	A	Synonymous SNV
17	orf02541	Ig-like domain-containing protein	2 651 078	Exonic	A	G	Synonymous SNV
18	orf02541	Ig-like domain-containing protein	2 651 117	Exonic	G	A	Synonymous SNV
19	orf02541	Ig-like domain-containing protein	2 651 159	Exonic	C	G	Synonymous SNV
20	orf02541	Ig-like domain-containing protein	2 651 160	Exonic	G	C	Nonsynonymous SNV
21	orf02541	Ig-like domain-containing protein	2 651 163	Exonic	T	−	Deletion
22	orf02541	Ig-like domain-containing protein	2 651 165	Exonic	G	T	Nonsynonymous SNV
23	orf02541	Ig-like domain-containing protein	2 651 166	Exonic	C	G	Nonsynonymous SNV
24	orf02541	Ig-like domain-containing protein	2 651 168	Exonic	−	A	Insertion
25	orf02541	Ig-like domain-containing protein	2 651 174	Exonic	A	G	Synonymous SNV
26	orf02541	Ig-like domain-containing protein	2 651 177	Exonic	C	G	Nonsynonymous SNV
27	orf02541	Ig-like domain-containing protein	2 651 180	Exonic	C	T	Synonymous SNV
28	orf02541	Ig-like domain-containing protein	2 651 181	Exonic	G	A	Nonsynonymous SNV
29	orf02541	Ig-like domain-containing protein	2 652 098	Exonic	G	A	Synonymous SNV
30	orf02541	Ig-like domain-containing protein	2 653 040	Exonic	A	G	Synonymous SNV
31	orf02542	Hypothetical protein	2 653 448	Exonic	G	−	Deletion
32	orf02804	Hypothetical protein	2 932 441	Downstream	C	−	Deletion
33	orf02913	Hypothetical protein	3 032 494	Exonic	T	C	Synonymous SNV
34	orf03675	Urease accessory protein ureD	3 867 248	Upstream	−	G	Insertion
35	orf03864	Alcohol dehydrogenase catalytic domain-containing protein	4 050 113	Upstream	T	G	Nonsynonymous SNV

Number	ORF number	Protein name	Locus	Block	Reference	Change	Type
− means there are no base pairs at this location.
1	orf00034	RNA-binding protein S1	37 964	Exonic	−	G	Insertion
2	orf00080	Hypothetical protein HALTITAN3299	86 078	Upstream	−	T	Insertion
3	orf00256	Lactoylglutathione lyase	257 737	Upstream	−	C	Insertion
4	orf00263	Hypothetical protein	261 715	Upstream	−	T	Insertion
5	orf00263	Hypothetical protein	261 744	Upstream	−	AG	Insertion
6	orf00469	Acetaldehyde dehydrogenase	471 178	Exonic	T	C	Synonymous SNV
7	orf00501	TRAP transporter substrate-binding protein	504 022	Upstream	−	G	Insertion
8	orf00566	Phosphopantothenoylcysteine decarboxylase	568 137	Exonic	C	A	Nonsynonymous SNV
9	orf00643	ABC transporter ATP-binding protein	647 351	Upstream	A	T	Synonymous SNV
10	orf00726	TRAP transporter large permease	732 078	Exonic	−	GT	Insertion
11	orf01257	UvrY/SirA/GacA family response regulator transcription factor	1 312 231	Upstream	−	A	Insertion
12	orf02141	Branched-chain amino acid ABC transporter permease	2 212 367	Exonic	−	C	Insertion
13	orf02185	Hypothetical protein	2 262 995	Upstream	−	G	Insertion
14	orf02433	Hypothetical protein	2 529 845	Exonic	−	T	Insertion
15	orf02522	Membrane proteins	2 628 488	Exonic	−	C	Insertion
16	orf02541	Ig-like domain-containing protein	2 650 751	Exonic	G	A	Synonymous SNV
17	orf02541	Ig-like domain-containing protein	2 651 078	Exonic	A	G	Synonymous SNV
18	orf02541	Ig-like domain-containing protein	2 651 117	Exonic	G	A	Synonymous SNV
19	orf02541	Ig-like domain-containing protein	2 651 159	Exonic	C	G	Synonymous SNV
20	orf02541	Ig-like domain-containing protein	2 651 160	Exonic	G	C	Nonsynonymous SNV
21	orf02541	Ig-like domain-containing protein	2 651 163	Exonic	T	−	Deletion
22	orf02541	Ig-like domain-containing protein	2 651 165	Exonic	G	T	Nonsynonymous SNV
23	orf02541	Ig-like domain-containing protein	2 651 166	Exonic	C	G	Nonsynonymous SNV
24	orf02541	Ig-like domain-containing protein	2 651 168	Exonic	−	A	Insertion
25	orf02541	Ig-like domain-containing protein	2 651 174	Exonic	A	G	Synonymous SNV
26	orf02541	Ig-like domain-containing protein	2 651 177	Exonic	C	G	Nonsynonymous SNV
27	orf02541	Ig-like domain-containing protein	2 651 180	Exonic	C	T	Synonymous SNV
28	orf02541	Ig-like domain-containing protein	2 651 181	Exonic	G	A	Nonsynonymous SNV
29	orf02541	Ig-like domain-containing protein	2 652 098	Exonic	G	A	Synonymous SNV
30	orf02541	Ig-like domain-containing protein	2 653 040	Exonic	A	G	Synonymous SNV
31	orf02542	Hypothetical protein	2 653 448	Exonic	G	−	Deletion
32	orf02804	Hypothetical protein	2 932 441	Downstream	C	−	Deletion
33	orf02913	Hypothetical protein	3 032 494	Exonic	T	C	Synonymous SNV
34	orf03675	Urease accessory protein ureD	3 867 248	Upstream	−	G	Insertion
35	orf03864	Alcohol dehydrogenase catalytic domain-containing protein	4 050 113	Upstream	T	G	Nonsynonymous SNV

Reference gene of XH26	Reference proteins of wild strain XH26 (NCBI identity, %)	NCBI accession number	Alter gene of G₉-72	Alter proteins of mutant strain G₉-72 (NCBI identity, %)	NCBI accession number
− means that no consistent protein sequence was found in the NCBI database.
orf00034	RNA-binding protein S1 (100.00)	WP030071723.1	cvfB	RNA-binding protein S1 (100.00)	WP038480019.1
orf00080	Hypothetical protein HALTITAN3299 (98.16)	ELY20074.1	orf00078	Hypothetical protein HALTITAN_3299 (98.16)	ELY20074.1
orf00257(glo1)	Lactoylglutathione lyase (100.00)	WP253486412.1	orf00223	DUF6164 family protein (100.00)	WP038480437.1
orf00263	Hypothetical protein (100.00)	WP038480446.1	orf00228	Hypothetical protein (100.00)	WP038480446.1
orf00501(yiaO)	TRAP transporter substrate-binding protein (100.00)	WP253486511.1	yiaO	TRAP transporter substrate-binding protein (100.00)	WP253486511.1
orf00726(siaT)	TRAP transporter large permease (99.28)	WP038481516.1	siaM	TRAP transporter large permease (93.63)	WP088700581.1
orf01257(gacA)	UvrY/SirA/GacA family response regulator transcription factor (100.00)	WP038482893.1	argF	Ornithine carbamoyltransferase (100.00)	WP198350143.1
orf02141	Branched-chain amino acid ABC transporter permease (100.00)	WP038485114.1	livH	Branched-chain amino acid ABC transporter permease (96.27)	WP088701692.1
orf02185	Hypothetical protein (100.00)	WP038485216.1	tonB	Energy transducer TonB (99.03)	WP271909853.1
orf02433	Hypothetical protein (100.00)	WP253485643.1	cphA	Hypothetical protein (100.00)	WP253485645.1
orf02541	Ig-like domain-containing protein (100.00)	WP253485764.1	orf02504	Ig-like domain-containing protein (99.70)	WP253485764.1
orf02542	Hypothetical protein (100.00)	WP253485765.1	orf02504	Ig-like domain-containing protein (99.70)	WP253485764.1
orf02804	Hypothetical protein (100.00)	WP167541166.1	orf02758	Restriction endonuclease, type Ⅰ, EcoR I, R subunit/Type Ⅲ (74.42)	AIA76090.1
orf03675(ureD)	Urease accessory protein ureD (100.00)	WP253486280.1	mntA	Manganese transporter (96.25)	AIA74233.1
orf00469(xylQ)	Aetaldehyde dehydrogenase (100.00)	WP038480889.1	xylQ	Acetaldehyde dehydrogenase (100.00)	AIA74850.1
orf00566	Phosphopantothenoylcysteine decarboxylase (100.00)	WP253486540.1	coaBC	Phosphopantothenoylcysteine decarboxylase (99.28)	AIA74921.1
orf00643(dppD)	ABC transporter ATP-binding protein (100.00)	WP231658237.1	orf00609	ABC transporter ATP-binding protein (−)	−
orf02913	Hypothetical protein (100.00)	WP038477584.1	orf02867	Hypothetical protein (71.36)	WP088698847.1
orf03864(rspB)	Alcohol dehydrogenase catalytic domain-containing protein (100.00)	WP253488167.1	rspB	Alcohol dehydrogenase catalytic domain-containing protein (98.70)	WP301271689.1

Reference gene of XH26	Reference proteins of wild strain XH26 (NCBI identity, %)	NCBI accession number	Alter gene of G₉-72	Alter proteins of mutant strain G₉-72 (NCBI identity, %)	NCBI accession number
− means that no consistent protein sequence was found in the NCBI database.
orf00034	RNA-binding protein S1 (100.00)	WP030071723.1	cvfB	RNA-binding protein S1 (100.00)	WP038480019.1
orf00080	Hypothetical protein HALTITAN3299 (98.16)	ELY20074.1	orf00078	Hypothetical protein HALTITAN_3299 (98.16)	ELY20074.1
orf00257(glo1)	Lactoylglutathione lyase (100.00)	WP253486412.1	orf00223	DUF6164 family protein (100.00)	WP038480437.1
orf00263	Hypothetical protein (100.00)	WP038480446.1	orf00228	Hypothetical protein (100.00)	WP038480446.1
orf00501(yiaO)	TRAP transporter substrate-binding protein (100.00)	WP253486511.1	yiaO	TRAP transporter substrate-binding protein (100.00)	WP253486511.1
orf00726(siaT)	TRAP transporter large permease (99.28)	WP038481516.1	siaM	TRAP transporter large permease (93.63)	WP088700581.1
orf01257(gacA)	UvrY/SirA/GacA family response regulator transcription factor (100.00)	WP038482893.1	argF	Ornithine carbamoyltransferase (100.00)	WP198350143.1
orf02141	Branched-chain amino acid ABC transporter permease (100.00)	WP038485114.1	livH	Branched-chain amino acid ABC transporter permease (96.27)	WP088701692.1
orf02185	Hypothetical protein (100.00)	WP038485216.1	tonB	Energy transducer TonB (99.03)	WP271909853.1
orf02433	Hypothetical protein (100.00)	WP253485643.1	cphA	Hypothetical protein (100.00)	WP253485645.1
orf02541	Ig-like domain-containing protein (100.00)	WP253485764.1	orf02504	Ig-like domain-containing protein (99.70)	WP253485764.1
orf02542	Hypothetical protein (100.00)	WP253485765.1	orf02504	Ig-like domain-containing protein (99.70)	WP253485764.1
orf02804	Hypothetical protein (100.00)	WP167541166.1	orf02758	Restriction endonuclease, type Ⅰ, EcoR I, R subunit/Type Ⅲ (74.42)	AIA76090.1
orf03675(ureD)	Urease accessory protein ureD (100.00)	WP253486280.1	mntA	Manganese transporter (96.25)	AIA74233.1
orf00469(xylQ)	Aetaldehyde dehydrogenase (100.00)	WP038480889.1	xylQ	Acetaldehyde dehydrogenase (100.00)	AIA74850.1
orf00566	Phosphopantothenoylcysteine decarboxylase (100.00)	WP253486540.1	coaBC	Phosphopantothenoylcysteine decarboxylase (99.28)	AIA74921.1
orf00643(dppD)	ABC transporter ATP-binding protein (100.00)	WP231658237.1	orf00609	ABC transporter ATP-binding protein (−)	−
orf02913	Hypothetical protein (100.00)	WP038477584.1	orf02867	Hypothetical protein (71.36)	WP088698847.1
orf03864(rspB)	Alcohol dehydrogenase catalytic domain-containing protein (100.00)	WP253488167.1	rspB	Alcohol dehydrogenase catalytic domain-containing protein (98.70)	WP301271689.1

紫外突变型盐单胞菌株的基因突变位点与四氢嘧啶高产的分子变异机制

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薛彬娟 ¹ , 韩睿 ² , 乔丽娟 ¹ , 李永臻 ¹ , 邢江娃 ¹ , 王嵘 ¹ , 沈国平 ¹^,^* , 朱德锐 ¹

微生物学报 | 研究报告 2024,64(12): 4902-4917

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微生物学报 | 研究报告 2024, 64(12): 4902-4917

紫外突变型盐单胞菌株的基因突变位点与四氢嘧啶高产的分子变异机制

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薛彬娟¹, 韩睿², 乔丽娟¹, 李永臻¹, 邢江娃¹, 王嵘¹, 沈国平¹^,^*, 朱德锐¹

作者信息

1 青海大学医学院, 基础医学研究中心, 青海西宁 810016

2 青海大学农林科学院, 蔬菜遗传与生理重点实验室, 青海西宁 810016

Mutation sites and high ectoine production mechanism of a Halomonas mutant induced by ultraviolet radiation

Binjuan XUE¹, Rui HAN², Lijuan QIAO¹, Yongzhen LI¹, Jiangwa XING¹, Rong WANG¹, Guoping SHEN¹^,^*, Derui ZHU¹

Affiliations

1 Department of Basic Medical Sciences, Medical College, Qinghai University, Xining 810016, Qinghai, China

2 Key Laboratory of Vegetable Genetics and Physiology, Academy of Agriculture and Forestry Sciences, Qinghai University, Xining 810016, Qinghai, China

出版时间: 2024-09-23 doi: 10.13343/j.cnki.wsxb.20240458

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摘要

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利用紫外诱变获得的一株高产四氢嘧啶(ectoine)的突变型坎帕尼亚盐单胞菌(Halomonas campaniensis) G₉-72，其突变位点、分子变异和高产四氢嘧啶的机制未知。【目的】探讨野生型菌株XH26与突变菌株G₉-72的突变位点与分子遗传变异机制，明确四氢嘧啶积聚量暴发的可能原因。【方法】采用PacBio Sequel Ⅱ平台进行全基因组测序，分析突变菌株的突变基因位点，结合氨基酸代谢通路分析突变基因与四氢嘧啶合成代谢的关联性，并进行RT-PCR验证。【结果】全基因组测序结果显示野生菌株XH26的基因组4.11 Mb，编码基因3 927个。突变菌株G₉-72的基因组存在35个突变位点，包括18个单核苷酸多态性突变、14个插入突变和3个缺失突变。代谢通路的关联分析显示：突变基因argF、coaBC和livH分别编码鸟氨酸氨甲酰基转移酶(NCBI数据库蛋白ArgF相似度100.00%)、磷酸泛酰半胱氨酸脱羧酶(蛋白CoaBC相似度99.28%)和支链氨基酸ABC转运体渗透酶(与蛋白LivH相似度96.27%)，分别参与延胡索酸、柠檬酸的合成以及增加支链氨基酸的吸收转运。上游代谢物的流量增加可能是突变菌株四氢嘧啶积聚量暴发的关键原因。RT-PCR验证四氢嘧啶代谢通路相关的20个基因，转录表达水平与预期分析相一致。【结论】突变基因argF、coaBC和livH的过表达增强四氢嘧啶合成的代谢流，与突变菌株四氢嘧啶积聚量的暴发有关，此为后续突变菌株酶分子的反应机制研究和发酵生产提供参考依据。

关键词

盐单胞菌 / 紫外诱变 / 突变基因 / 比较基因组学 / 四氢嘧啶

Abstract

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A mutant (G₉-72) of Halomonas campaniensis exhibiting high ectoine production was obtained by ultraviolet (UV) mutagenesis. The mutation sites, molecular variations, and high ectoine production mechanism of this mutant remain unknown.[Objective] To investigate the mutation sites and genetic variations of G₉-72 compared with the wild type strain XH26 and identify the potential causes of ectoine accumulation outbreak. [Methods] PacBio Sequel Ⅱ was used for whole-genome sequencing, and the mutation sites in the genome of the mutant were identified based on the sequencing results. The amino acid metabolic pathways were analyzed to reveal the association between mutated genes and ectoine synthesis, and the results were verified by RT-PCR. [Results] The genome of strain XH26 was 4.11 Mb, encoding 3 927 genes. Compared with strain XH26, G₉-72 showed 35 mutation sites, including 18 single nucleotide polymorphism mutations, 14 insertion mutations, and 3 deletion mutations. The mutated genes argF, coaBC, and livH, which encoded ornithine transcarbamylase (100.00% similarity with ArgF proteins in NCBI database), phosphopantothenoylcysteine decarboxylase (99.28% similarity with CoaBC proteins in NCBI database), and branched-chain amino acid ABC transporter permease (96.27% similarity with LivH proteins in NCBI database), were implicated in the synthesis of fumaric acid, citric acid and the absorption and transport of branched-chain amino acids, respectively. The increased flow of upstream metabolites may be the key reason for the sharply increased accumulation of ectoine in the mutant. RT-PCR verified 20 genes related to the ectoine metabolic pathway, and the transcriptional expression levels were consistent with the expected analysis. [Conclusion] The overexpression of genes argF, coaBC, and livH enhanced the anabolic flow of ectoine, which contributed to a significant increase in ectoine accumulation in the mutant. This finding provides a reference point for subsequent studies on the reaction mechanisms of enzymes in the mutant and the fermentation production.

Key words

Halomonas / ultraviolet mutagenesis / mutation gene / comparative genomics / ectoine

引用本文

薛彬娟, 韩睿, 乔丽娟, 李永臻, 邢江娃, 王嵘, 沈国平, 朱德锐. 紫外突变型盐单胞菌株的基因突变位点与四氢嘧啶高产的分子变异机制. 微生物学报, 2024 , 64 (12) : 4902 -4917 . DOI: 10.13343/j.cnki.wsxb.20240458

Binjuan XUE, Rui HAN, Lijuan QIAO, Yongzhen LI, Jiangwa XING, Rong WANG, Guoping SHEN, Derui ZHU. Mutation sites and high ectoine production mechanism of a Halomonas mutant induced by ultraviolet radiation[J]. Acta Microbiologica Sinica, 2024 , 64 (12) : 4902 -4917 . DOI: 10.13343/j.cnki.wsxb.20240458

正文

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四氢嘧啶(ectoine)是中度嗜盐菌或耐盐菌胞内积聚的相容溶质，作为细胞的保护剂和稳定剂，已被广泛应用于化妆品、生物制剂、药物佐剂等研究领域^[1-2]。四氢嘧啶的合成途径以天冬氨酸半醛(l-aspartate-4-semialdehyde, ASA)为前体底物，经二氨基丁酸转氨酶(diaminobutyrate transaminase, EctB)、二氨基丁酸乙酰基转氨酶(diaminobutyrate acetyl transferase, EctA)和四氢嘧啶合成酶(ectoine synthase, EctC) 3步催化合成^[3]。然而，四氢嘧啶的化学合成工艺较为复杂，副产物多，且能耗大，易造成环境污染。国内外学者采用多种四氢嘧啶过量化生产的策略，以提高菌株胞内的四氢嘧啶积累，如物理/化学诱变、发酵工程法、基因工程或系统代谢工程(碳/氮代谢流)等^[4-5]。王冠凤等^[6]利用海神盐单胞菌(Halomonas neptunia)进行紫外线(ultraviolet, UV)和亚硝基胍的复合诱变，筛选获得突变株UN2，四氢嘧啶的摇瓶发酵产量达1.80 g/L (提高53.80%)。Schiraldi等^[7]利用嗜盐海球菌(Marinococcus halophilus)进行批式和补料分批发酵，四氢嘧啶产量分别为1.60 g/L和3.60 g/L。Chen等^[8]将延长盐单胞菌(H. elongata)的四氢嘧啶合成基因簇ectABC成功导入大肠杆菌(Escherichia coli) MG1655，构建四氢嘧啶异源合成工程菌，并敲除lysA基因以削弱赖氨酸(lysine, Lys)的合成代谢分流，最终四氢嘧啶的产量达12.70 g/L (提高16.85倍)。Ma等^[9]对蓝晶盐单胞菌(H. bluephagenesis) TD01中3个基因簇ectABC、lysC和asd过表达，并通过CRISPR/Cas9基因编辑工具敲除基因doeA和ectD，防止四氢嘧啶降解，获得重组菌株TD-ADEL-58，分批补料生产四氢嘧啶，28 h产量达28 g/L。

课题组前期以四氢嘧啶产量491.19 mg/L的野生坎帕尼亚盐单胞菌(Halomonas campaniensis) XH26为研究材料，经多轮紫外循环诱变和筛选，获得遗传稳定的突变菌株G₈-52和G₉-72^[10]。比较基因组学分析野生菌株XH26和突变菌株G₈-52 (四氢嘧啶产量1.51 g/L)，结果显示菌株G₈-52存在24个基因突变，其中基因orf00723和orf02403 (lipA)分别突变编码γ-氨基丁酸转移酶(DavT)和NAD-琥珀酸半醛脱羧酶(GabD)，推测四氢嘧啶的产量增加可能与琥珀酸(succinic acid)的合成代谢流有关^[11]。然而，突变菌株G₉-72的四氢嘧啶产量更高，达到(1.93±0.01) g/L，何种突变基因参与四氢嘧啶合成代谢的网络通路调控，致使四氢嘧啶积聚量暴发，有待深入探讨。因此，本研究采用PacBio Sequel Ⅱ平台进行野生菌株XH26和突变菌株G₉-72的全基因组测序，利用比较基因组学分析关键的突变基因和可能涉及的代谢通路，以此探究四氢嘧啶积聚量的暴发原因，为后续突变菌株的发酵生产和实际应用提供一定的理论参考。

1 材料与方法

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1.1 菌株和培养基

野生菌株H. campaniensis sp. XH26 (CCTCCM 2019776M)和紫外突变菌株G₉-72，均保存于青海大学医学院基础医学研究中心。菌株培养基(g/L, pH 8.0)^[10]：NaCl 58.50，KCl 55.88，MgSO₄‧7H₂O 24.65，l-谷氨酸钠(monosodium glutamate monohydrate, MSG) 6.50，柠檬酸钠3.00，酶水解酪素7.50，无水CaCl₂ 0.20，酵母抽提物2.00，121 ℃灭菌15 min。

1.2 主要试剂和仪器

分析纯NaCl、MSG、无水CaCl₂、MgSO₄‧7H₂O、KCl、琼脂、柠檬酸钠和酶水解酪素等，北京索莱宝科技有限公司；戊二醛固定液，南京森贝伽生物科技有限公司；乙腈、色谱柱和DNA-free^TM DNA去除试剂盒，赛默飞世尔科技公司；微孔过滤器，天津市津腾实验设备有限公司；SPARKeasy细菌基因组DNA快速提取试剂盒，山东思科捷生物技术有限公司；TransZol Up强化RNA提取试剂盒，北京全式金生物技术股份有限公司；PrimeScript^TM RT reagent Kit with gDNA Eraser和TB Green Premix Ex Taq^TM Ⅱ，TaKaRa公司。

恒温培养摇床，上海一恒科学仪器有限公司；高效液相色谱仪，Agilent公司；可见分光光度计，上海舜宇恒平科学仪器公司；TGrinder第三代高速组织研磨器，天根生化科技(北京)有限公司；扫描电子显微镜，FEI公司；高通量测序平台，PacBio公司；PCR仪，Bio-Rad公司；实时荧光定量PCR仪，Roche公司。

1.3 菌株形态学分析

活化菌液按1%比例接种于100 mL的液体培养基，37 ℃、180 r/min培养12 h至对数生长期(OD₆₀₀约为1.20)，再利用平板划线观察菌落形态(37 ℃培养36 h)。液体培养的菌悬液转移至1.5 mL尖底离心管中，8 000 r/min离心5 min，弃上清液，用吸管沿管壁缓慢加入3%戊二醛固定液，吹打混匀、悬浮固定细菌，制备扫描电镜样品，观察菌体形态。

1.4 菌株生长和四氢嘧啶检测

活化菌液(OD₆₀₀约为1.20)按1%的接种量分别转接至摇瓶内(n=3)，37 ℃、180 r/min培养60 h，其间每4 h测定菌液光度密度值(OD₆₀₀)，并提取ectoine进行HPLC定量分析^[12]。HPLC检测条件：流动相乙腈/水=80/20 (体积比)，流速1.0 mL/min，柱温30 ℃，检测波长210 nm，进样量5 µL。最后以培养时间(t)为横坐标，OD₆₀₀值与四氢嘧啶产量为纵坐标，绘制菌株生长和四氢嘧啶产量的关系曲线。

1.5 全基因组测序及基础分析

采用细菌基因组DNA试剂盒进行DNA抽提，利用琼脂糖凝胶电泳法和微量分光光度计检测基因组DNA的完整度、纯度(满足OD₂₆₀/OD₂₈₀= 1.8−2.0，OD₂₆₀/OD₂₃₀=2.0−2.2)和浓度(> 50 ng/µL)。检测合格的DNA样本进行文库构建，利用PacBio Sequel Ⅱ平台进行测序分析，由武汉菲沙基因信息公司完成。利用Gapclose软件(v1.12)进行原始数据预处理和冗余序列去除，过滤序列采用HGAP软件(v4.0)组装。分别利用Glimmer软件(v3.02)、tRNAscan-SE软件(v2.0)和RNAmmer软件(v1.2)进行编码mRNA、tRNA和rRNA基因预测。通过基因本体论(gene ontology, GO)数据库(http://geneontology.org/)和京都基因和基因组百科全书(Kyoto encyclopedia of genes and genomes, KEGG)数据库(http://www.genome.jp/kegg/)分别进行基因序列比对和功能注释。

1.6 突变位点与代谢通路关联分析

利用Mummer软件(v4.0)进行比较基因组分析，参考基因组为野生菌株XH26，查询基因组为突变菌株G₉-72，采用Delta-filter工具进行比对过滤，以获得有效突变信息。利用Show-snps程序进行全基因组单核苷酸多态性(single nucleotide polymorphism, SNP)和插入/缺失(insertion-deletion, InDel)检测，并使用ANNOVAR软件(v20191024)进行SNP和InDel注释；然后利用NCBI-BLAST在线程序(http://www.ncbi.nlm.nih.gov/BLAST/)比对分析基因的突变位点，并结合KEGG分析突变基因与四氢嘧啶代谢通路的关联性。

1.7 关键基因RT-PCR检测

采用RNA提取试剂盒提取菌株总RNA (n=3)，利用DNA去除试剂盒去除样本残留的DNA，并通过PCR仪和NanoDrop 2000检测DNA去除、RNA纯度(OD₂₆₀/OD₂₈₀=1.7−2.0)和浓度(浓度500−700 ng/µL)。符合要求的RNA使用反转录试剂逆转录得到cDNA (−80 ℃保存)。按RT-PCR试剂盒进行定量检测，反应体系20 µL：TB溶液10 µL，正、反向引物(10 µmol/L)各0.8 µL，无菌无酶水4.4 µL，cDNA 4 µL。RT-PCR反应条件：95 ℃预变性3 min；95 ℃变性10 s，58 ℃退火20 s，72 ℃延伸30 s，45个循环。以GAPDH作为内参基因，利用2^−ΔΔC_t法比较同一基因在两菌株中的相对表达水平。本研究中所有的引物由生工生物工程(上海)股份有限公司合成(表1)。

2 结果与分析

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2.1 野生菌株和突变菌株的形态学与生长特性比较分析

野生菌株XH26菌落(图1A)与突变菌株G₉-72菌落(图1D)形态基本一致，菌落圆形、乳白色、不透明、轻微隆起，且边缘规则，但突变菌落的直径略有变小。电镜形态显示，两株菌体均呈短杆状，略微弯曲，两端圆润，无鞭毛。野生菌株XH26的大小为(1.66−5.47) µm× (0.60−0.77) µm (图1B)；而突变菌株G₉-72较短宽，大小为(1.57−2.79) μm×(0.76−0.99) µm (图1E)。菌株生长特性分析显示，野生菌株XH26 (图1C)和突变菌株G₉-72 (图1F)均在4 h时进入对数生长期。野生菌株XH26在16 h左右结束对数生长期，生长速度逐渐变得缓慢，32 h时胞内四氢嘧啶积聚量可达峰值0.64 g/L；突变菌株G₉-72在20 h左右结束对数生长期，但随着培养时间的延长，四氢嘧啶产量仍然能持续累积，52 h达到峰值1.93 g/L。由此表明，突变菌株G₉-72进入对数生长期的时间更长，且四氢嘧啶的持续合成能力较好。

2.2 全基因组测序基础分析

利用PacBio平台进行野生菌株XH26和突变菌株G₉-72的全基因组测序，并对测序结果进行基础分析(表2)。结果显示，野生菌株XH26和突变菌株G₉-72的全基因组大小分别为4.11 Mb (G+C含量52.62%)和4.06 Mb (G+C含量52.55%)。菌株XH26和突变菌株G₉-72基因组预测到的编码基因分别为3 927个和3 882个，其中蛋白质编码基因各有3 830个和3 785个；两株菌的tRNA编码基因(63个)和rRNA编码基因(18个)的数量相等。

2.3 GO/KEGG注释与聚类分析

基于GO功能数据库比对分析野生菌株XH26和突变菌株G₉-72基因的编码产物，可能涉及参与的生物过程、分子功能以及细胞组成(图2A)。结果显示，野生菌株XH26和突变菌株G₉-72的基因组分别注释到2 618个和2 592个预测基因。其中，生物学过程亚类中的预测基因主要富集于代谢过程(分别为1 466个和1 447个)和细胞代谢过程(分别为1 297个和1 279个)；细胞组分亚类中的预测基因主要集中于细胞(分别为733个和727个)和细胞组分(分别为762个和754个)；分子功能亚类中的预测基因主要富集在催化功能(分别为1 495个和1 481个)及锚定/结合作用(分别为1 108个和1 098个)。

利用KEGG数据库分析野生菌株XH26和突变菌株G₉-72的基因表达产物，并进行可能参与的代谢通路统计分析(图2B)。结果显示，野生菌株XH26和突变菌株G₉-72的KEGG代谢通路分别注释到2 305个和2 272个蛋白质。大多数注释的通路蛋白聚类于氨基酸代谢(分别为277个和276个)、碳水化合物代谢(221个和220个)、辅助因子和维生素代谢(分别为182个和181个)、跨膜转运(分别为178个和177个)以及能量代谢(分别为140个和139个)等。在次级代谢物的生物合成途径中，野生菌株XH26和突变菌株G₉-72分别有44个和43个注释基因(或蛋白质)。

2.4 比较基因组学分析突变位点

基于基因组两两比对的突变信息集，采用Show-snps程序和ANNOVAR软件分别进行SNP和InDel检测和功能注释(表3)。结果显示，与野生菌株XH26相比，突变菌株G₉-72存在小片段序列突变的数目为17个(包括14个插入和3个缺失突变)，单核苷酸多态性(SNP)数目为18个(包括11个同义突变和7个非同义突变)，共计35个突变位点。其中24个突变发生在结构基因内部，10个突变位于结构基因上游区域，以及1个突变位于结构基因下游区域。

2.5 突变基因功能解析

深入分析24个结构基因及翻译蛋白质的突变位点，进行突变前后的功能比较(表4)。结果显示，突变蛋白主要包括RNA结合蛋白(由基因cvfB编码)、DUF6164家族蛋白(基因orf00223编码)、3个假定蛋白(基因orf00228、cphA与orf02867编码)、TRAP转运体底物结合蛋白(基因yiaO编码)、TRAP转运体大渗透酶(基因siaM编码)、鸟氨酸氨甲酰基转移酶(基因argF编码)、支链氨基酸ABC转运体渗透酶(基因livH编码)、TonB家族转录调节因子(基因tonB编码)、类Ig结构域蛋白(基因orf02504编码)、限制性内切酶(基因orf02758编码)、锰转运体(基因mntA编码)、乙醛脱氢酶(基因xylQ编码)、磷酸泛酰半胱氨酸脱羧酶(基因coaBC编码)、ABC转运蛋白(基因orf00609编码)以及醇脱氢酶的催化结构域蛋白(基因rspB编码)。

突变菌株G₉-72中存在3个关键的突变蛋白(ArgF、CoaBC和LivH)，可能增加三羧酸循环(tricarboxylic acid cycle, TCA)中延胡索酸(fumaric acid)、柠檬酸(citric acid)、乙酰辅酶A (acetyl CoA)和琥珀酰辅酶A (succinyl CoA) 的碳/氮代谢流，从而提高四氢嘧啶的合成量。首先，突变菌株G₉-72的基因orf01257 (gacA)发生插入突变，框移突变导致翻译表达为鸟氨酸氨甲酰基转移酶(ornithine transcarbamylase)，与NCBI数据库中的蛋白ArgF同源(相似度100.00%)。KEGG代谢通路分析显示，胞内的N-乙酰鸟氨酸(N-acetyl-omithine)依次在酶ArgF、ArgE、ArgG和ArgH作用下可生成延胡索酸。其次，突变菌株G₉-72的基因orf00566发生点突变，翻译表达磷酸泛酰半胱氨酸脱羧酶(phosphopantothenoyl cysteine decarboxylase)，与NCBI数据库中的蛋白CoaBC同源(相似度99.28%)。以半胱氨酸(cysteine, Cys)为底物，经系列酶CoaBC、CoaD、CoaE和CoaA催化生成乙酰CoA，再经柠檬酸合酶(基因gltA)缩合生成柠檬酸。再次，突变菌株G₉-72的基因orf02141发生插入突变，框移突变导致翻译表达为支链氨基酸ABC转运体渗透酶(branched-chain amino acid ABC transporter permease)，与NCBI数据库中的蛋白LivH同源(相似度96.27%)。蛋白LivH参与支链氨基酸的跨膜转运，将胞外的亮氨酸(leucine, Leu)、异亮氨酸(isoleucine, Ile)和组氨酸(histidine, His)转运至胞内。

2.6 突变基因差异表达验证

以筛选到的3个突变基因(argF、coaBC和livH)为参考，关联分析上下游的代谢通路和甄选可能参与的关键基因，进行RT-PCR转录差异分析(图3)。结果显示，与野生菌株XH26相比，突变菌株G₉-72中除基因ectD外，其他基因均转录表达上调(P < 0.000 1)。首先，基因argA、argB、argC、argD、argE、argF、argG、argH表达上调可增强谷氨酸(glutamate, Glu)合成延胡索酸途径，从而提高草酰乙酸(oxaloacetic acid, OAA)和天冬氨酸(aspartate, Asp)的代谢流。其次，基因coaBC、coaD、coaE、gltA转录表达上调，也可增加TCA中OAA代谢流量促进Asp的转化代谢。再次，基因livH转录的表达量升高，说明进入胞内的His、Leu和Ile量增加，后续Leu与Ile的分解代谢生成乙酰CoA，His则可合成Asp，以此增强四氢嘧啶合成代谢流，致使胞内四氢嘧啶的合成量升高。最后，合成四氢嘧啶的主要基因lysC、asd、ectA、ectB、ectC表达量均上调，提升了四氢嘧啶的直接合成量；同时，编码四氢嘧啶羟化酶(ectoine hydroxylase)的基因ectD转录表达下调，减少了四氢嘧啶向羟基四氢嘧啶(5-hydroxyectoine)的代谢转化。

3 讨论

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3.1 紫外突变位点与四氢嘧啶暴发的关联性分析

目前，微生物诱变育种主要包括紫外诱变、化学诱变和常压室温等离子体诱变等方法。紫外诱变技术因操作简便、成本低、遗传稳定性良好，适用于实验室突变菌株的大规模筛选^[12-15]。紫外诱变常选取的最佳光源为短波紫外线(波长200−280 nm)，经短波紫外线辐射后易形成嘧啶二聚体，阻碍碱基间的正常配对，造成基因复制的错配，从而导致突变菌株发生形态、生长特性的变化^[16-19]。本研究采用短波紫外线(25 W，30 cm，诱变50 s)诱变盐单胞菌XH26，获得一株高产四氢嘧啶的突变菌株G₉-72 (四氢嘧啶产量1.93 g/L，提高2.97倍)。与野生菌株相比，突变菌株的菌落形态基本一致，但菌落直径更小，生长略微缓慢，菌体形态略微短宽。盐单胞菌胞内的四氢嘧啶积累是以ASA为底物，依次在酶EctB、EctA和EctC的作用下逐步合成(图3B)^[3]。我们的前期研究发现，盐单胞菌XH26的四氢嘧啶生物合成与Asp和ASA代谢直接相关，与Glu、谷氨酰胺(glutamine, Gln)、半胱氨酸以及TCA中的琥珀酸、苹果酸(malic acid)、OAA等间接相关^[20-22]。此外，四氢嘧啶的合成代谢通路中还存在代谢分流支路，如ASA经高丝氨酸脱氢酶(基因hom编码)作用可分流合成甜菜碱(betaine)；又如代谢中间物天冬氨酰磷酸(l-4-aspartylphosphate)由4-羟基-四氢二吡啶甲酸合成酶(基因dapA编码)的催化作用，可生成赖氨酸^[23-25]。

课题组前期对获得的另一遗传稳定的突变菌株G₈-52进行比较基因组学分析^[11]，发现紫外突变菌株G₈-52存在2个关键突变酶：γ-氨基丁酸转移酶(基因davT)和NAD-琥珀酸半缩醛脱羧酶(基因gabD)，催化反应增强了Glu向琥珀酸半缩醛(succinic semialdehyde)和延胡索酸的转化代谢，从而提高OAA、Asp和四氢嘧啶的代谢流。本研究中，与野生菌株XH26相比，突变菌株G₉-72筛选出的2个突变基因argF和coaBC，分别表达鸟氨酸氨甲酰基转移酶和磷酸泛酰半胱氨酸脱羧酶(图3B)；二者与四氢嘧啶的合成代谢通路密切关联，分别促进延胡索酸和柠檬酸的合成。通过增加TCA的代谢流量促进Asp的转化代谢，可能是导致四氢嘧啶产量暴发的主要原因。至此，无论是突变菌株G₉-72还是菌株G₈-52，四氢嘧啶合成量的暴发机制均是通过Glu→TCA→OAA→Asp→四氢嘧啶代谢通路介导实现，增强其中某一物质的代谢流，最终促进Asp转化合成四氢嘧啶。

此外，本研究中突变菌株G₉-72的部分ABC转运蛋白和TRAP转运体的编码基因发生突变(表4)，如TRAP转运体底物结合蛋白(基因yiaO编码)、TRAP转运体大渗透酶(基因siaM编码)、支链氨基酸ABC转运体渗透酶(基因livH编码)和ABC转运蛋白(基因orf00609编码)。ABC转运蛋白主要参与糖类、氨基酸、蛋白质及代谢产物等物质的跨膜运输和四氢嘧啶的吸收和排泄，而TRAP转运体(tripartite ATP-independent periplasmic transporter)与ABC转运系统(ATP-binding cassette transporters)具有相似的胞外溶质结合受体，与ABC转运蛋白功能相似^[26-27]。其中，支链氨基酸ABC转运体渗透酶可将His、Leu和Ile从胞外转运至胞内，可能增加Asp合成四氢嘧啶的代谢流量。由此我们推测，基因livH的突变可能是导致四氢嘧啶积聚量暴发的次要原因。

3.2 突变基因遗传改造和代谢工程的应用前景

代谢工程是借助基因工程技术有目的的实施细胞代谢途径的修饰与改造，以增加目标产物的合成^[28]。截至目前，利用系统代谢工程或合成生物学的技术策略来促进四氢嘧啶的生物合成，已成为当前的研究热点。首先，基于四氢嘧啶代谢通路，采用过表达四氢嘧啶合成途径的相关代谢酶基因，以增强中间代谢物或合成前体的供应流。如过表达天冬氨酸转氨酶基因(aspC)、天冬氨酸激酶基因(lysC)^[29]和天冬氨酸半醛脱氢酶基因(asd)^[30]以增强Asp的代谢流；过表达丙酮酸激酶基因(pk)^[31]以增强OAA的代谢流；过表达谷氨酸脱氢酶基因(gdh)以增强二氨基丁酸(l-diaminobutyric acid)的代谢流^[32]。其次，利用CRISPR/Cas9基因编辑工具敲除四氢嘧啶合成代谢分流或降解的相关基因，构建代谢分流缺陷型菌株。如设计敲除甜菜碱合成基因hom^[33]、赖氨酸合成基因dapA、羟基四氢嘧啶合成基因ectD和四氢嘧啶水解的相关酶基因doeA/B/C^[9]，以减少四氢嘧啶合成代谢流的分流损失。再次，挖掘新型的代谢调控基因或上游关键代谢的酶基因，采用分子克隆技术进行系统代谢工程整合或改造，实现四氢嘧啶合成代谢通路的优化改造和系统整合，提高代谢中间物(或前体)的流量和四氢嘧啶的合成量。如整合表达突变菌株G₈-52的关键突变基因davT和gabD，以及本研究菌株G₉-72的潜在突变基因argF、coaBC和livH，以增加四氢嘧啶合成上游底物的代谢流；又如构建重组整合质粒(argA/B/C/D/E/F/G/H+mdh)或(coaBC/D/E+gltA)，并利用T₇强启动子诱导基因表达，以增强上游的OAA代谢流；同时，过表达基因aspC、lysC、asd和ectABC基因簇，进一步提高四氢嘧啶的合成量。最后，利用合成生物学和AI (artificial intelligence)智能设计，以简化的“细胞工厂”为载体，运用网络代谢重构技术，精准调控天然次级代谢物的碳/氮源代谢流，可能实现ectoine的单通路发酵生产^[34]。

4 结论

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盐单胞菌株XH26突变前后的菌落形态基本一致，但突变菌株的菌体略微短宽，且四氢嘧啶的积聚量提升2.97倍(为1.93 g/L)。基于比较基因组学分析发现，突变菌株G₉-72存在35个基因突变位点，包括18个单核苷酸多态性位点，17个碱基插入/缺失突变。关键突变基因argF和coaBC分别编码鸟氨酸氨甲酰基转移酶和磷酸泛酰半胱氨酸脱羧酶，可能增加TCA中延胡索酸和柠檬酸的代谢流，以此增加四氢嘧啶合成的代谢流，这可能是菌株G₉-72四氢嘧啶积聚量暴发的关键原因。关键突变基因livH编码支链氨基酸ABC转运体渗透酶，促进胞外His、支链氨基酸Leu和Ile转运至胞内，增加Asp合成四氢嘧啶的代谢流，可能是导致四氢嘧啶积聚量暴发的次要原因。后续我们将采用分子对接和分子动力学模拟，并结合体外酶促反应实验，从酶分子结构、功能和催化效能的角度进一步解析突变菌株高效积聚四氢嘧啶的作用机制。

基金

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国家自然科学基金(32260019)
青海中央引导地方科技发展资金项目(2024ZY015)

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2024年第64卷第12期

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文章信息

doi: 10.13343/j.cnki.wsxb.20240458

接收时间：2024-07-24
首发时间：2026-03-21
出版时间：2024-09-23

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出版历史

收稿日期：2024-07-24
录用日期：2024-09-19

基金

National Natural Science Foundation of China(32260019)

国家自然科学基金(32260019)

Qinghai Central Government Guide Local Science and Technology Development Fund Project(2024ZY015)

青海中央引导地方科技发展资金项目(2024ZY015)

作者信息

1 青海大学医学院, 基础医学研究中心, 青海西宁 810016

2 青海大学农林科学院, 蔬菜遗传与生理重点实验室, 青海西宁 810016

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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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Genes name	Primer sequences (5′→3′)
Amino-acid-N-acetyltransferase (argA)	F: CACGGTGTAGAGCTTAGCGT; R: CAGCATGTTGTGCGACTTCC
N-acetylglutamate kinase (argB)	F: GTGCGGTCATCTCAGGACTT; R: CTTTGTGAACGGCATGCGAG
N-acetyl-γ-glutamyl-phosphate reductase (argC)	F: CTTTGACGCTGCGAGTTTCC; R: CCAGCGAATCGATGAAAGCC
Aspartate aminotransferase (argD)	F: GAAAGTGCGGCCATGGAAAG; R: CACTGAGCGTACTTTTGCCG
N-acetylornithine deacetylase (argE)	F: CGTTGAAATCACCACCGAGC; R: TGAGGTGTTTCTAGCGCCTG
Ornithine transcarbamylase (argF)	F: CGTTAATCACAGGCACGCTG; R: AGAGCCGATTGAAGACACCG
Argininosuccinate synthase (argG)	F: CGCTTAGCAATCAACGGACG; R: AGGTTGTGCTGGCGTATTCA
Argininosuccinate lyase (argH)	F: CACGCTCTTCATCGGTCAGT; R: AGCCAAGCTACGAACCAGTC
Phosphopantothenoylcysteine decarboxylase (coaBC)	F: GGCGTTGGTTAGTAGGCAGT; R: AAGCGGCCCAGTTAATCTCC
Pantetheine-phosphate adenylyltransferase (coaD)	F: CCTATCACCAACGGCCACTT; R: TCAAGACTAGGCTGCTTGCC
Dephospho-CoA kinase (coaE)	F: CCGAACACTTTGGCACAGAC; R: GTGCGTGATAATTGCAGCGA
Citrate synthase (gltA)	F: TGAGCCTGCTAAGCGTACTG; R: GTTCAGCAATCCGTGCGAAG
Malate dehydrogenase (mdh)	F: GTGCTTGCCGGTTTTCTGAG; R: GCGTGTACTCGTTGTCGGTA
Branched-chain amino acid transporter (livH)	F: ATGCCTCCTTACCGAACAGC; R: GGTGCGATTCTTACTGGCCT
Aspartate kinase (lysC)	F: GAGGACGCTATGGAAGAAC; R: GCGATAGGACCAAGAATACG
Aspartate-semialdehyde dehydrogenase (asd)	F: GAACGACAAAGACGCTACAG; R: CAACACTGAAGGCTGACAAG
Diaminobutyrate acetyl transferase (ectA)	F: AGTCGCTGATGCTGTGGTTGG; R: ACATCAAGCGGCGGACAAG
Diaminobutyrate transaminase (ectB)	F: TGGTATTGATGTTGTCTCTGG; R: TCACTACTTCGCCGTCTTGG
Ectoine synthase (ectC)	F: TGAAGGCGAAGGCGAAGTAG; R: GATGTTCGTCGTGCTGATCC
Ectoine hydroxylase (ectD)	F: CCAGAAAGCCACGATATTC; R: TGATGCACGTAAGGATTAGCG
Housekeeping gene (GAPDH)	F: TCCTTCCCTAAACTCGCACCT; R: ACGATAGTAGCGTCAGCAT

Genes name

Primer sequences (5′→3′)

Amino-acid-N-acetyltransferase (argA)

F: CACGGTGTAGAGCTTAGCGT; R: CAGCATGTTGTGCGACTTCC

N-acetylglutamate kinase (argB)

F: GTGCGGTCATCTCAGGACTT; R: CTTTGTGAACGGCATGCGAG

N-acetyl-γ-glutamyl-phosphate reductase (argC)

F: CTTTGACGCTGCGAGTTTCC; R: CCAGCGAATCGATGAAAGCC

Aspartate aminotransferase (argD)

F: GAAAGTGCGGCCATGGAAAG; R: CACTGAGCGTACTTTTGCCG

N-acetylornithine deacetylase (argE)

F: CGTTGAAATCACCACCGAGC; R: TGAGGTGTTTCTAGCGCCTG

Ornithine transcarbamylase (argF)

F: CGTTAATCACAGGCACGCTG; R: AGAGCCGATTGAAGACACCG

Argininosuccinate synthase (argG)

F: CGCTTAGCAATCAACGGACG; R: AGGTTGTGCTGGCGTATTCA

Argininosuccinate lyase (argH)

F: CACGCTCTTCATCGGTCAGT; R: AGCCAAGCTACGAACCAGTC

Phosphopantothenoylcysteine decarboxylase (coaBC)

F: GGCGTTGGTTAGTAGGCAGT; R: AAGCGGCCCAGTTAATCTCC

Pantetheine-phosphate adenylyltransferase (coaD)

F: CCTATCACCAACGGCCACTT; R: TCAAGACTAGGCTGCTTGCC

Dephospho-CoA kinase (coaE)

F: CCGAACACTTTGGCACAGAC; R: GTGCGTGATAATTGCAGCGA

Citrate synthase (gltA)

F: TGAGCCTGCTAAGCGTACTG; R: GTTCAGCAATCCGTGCGAAG

Malate dehydrogenase (mdh)

F: GTGCTTGCCGGTTTTCTGAG; R: GCGTGTACTCGTTGTCGGTA

Branched-chain amino acid transporter (livH)

F: ATGCCTCCTTACCGAACAGC; R: GGTGCGATTCTTACTGGCCT

Aspartate kinase (lysC)

F: GAGGACGCTATGGAAGAAC; R: GCGATAGGACCAAGAATACG

Aspartate-semialdehyde dehydrogenase (asd)

F: GAACGACAAAGACGCTACAG; R: CAACACTGAAGGCTGACAAG

Diaminobutyrate acetyl transferase (ectA)

F: AGTCGCTGATGCTGTGGTTGG; R: ACATCAAGCGGCGGACAAG

Diaminobutyrate transaminase (ectB)

F: TGGTATTGATGTTGTCTCTGG; R: TCACTACTTCGCCGTCTTGG

Ectoine synthase (ectC)

F: TGAAGGCGAAGGCGAAGTAG; R: GATGTTCGTCGTGCTGATCC

Ectoine hydroxylase (ectD)

F: CCAGAAAGCCACGATATTC; R: TGATGCACGTAAGGATTAGCG

Housekeeping gene (GAPDH)

F: TCCTTCCCTAAACTCGCACCT; R: ACGATAGTAGCGTCAGCAT

Analysis content	Wild strain XH26	Mutant strain G₉-72
Molecule shape	Circular	Circular/Oval
Coverage (%)	100	100
Genome size (bp)	4 112 053	4 058 888
G+C content (%)	52.62	52.55
Coding sequences (CDS)	104 708	126 226
Total RNA genes	97	97
tRNAs	63	63
5S rRNAs	6	6
16S rRNAs	6	6
23S rRNAs	6	6
Other RNA genes	16	16
Predicted coding genes	3 927	3 882
Total length of predicted coding genes (bp)	3 687 732	3 636 840

Analysis content

Wild strain XH26

Mutant strain G₉-72

Molecule shape

Circular

Circular/Oval

Coverage (%)

100

Genome size (bp)

4 112 053

4 058 888

G+C content (%)

52.62

52.55

Coding sequences (CDS)

104 708

126 226

Total RNA genes

tRNAs

5S rRNAs

16S rRNAs

23S rRNAs

Other RNA genes

Predicted coding genes

3 927

3 882

Total length of predicted coding genes (bp)

3 687 732

3 636 840

Number	ORF number	Protein name	Locus	Block	Reference	Change	Type
− means there are no base pairs at this location.
1	orf00034	RNA-binding protein S1	37 964	Exonic	−	G	Insertion
2	orf00080	Hypothetical protein HALTITAN3299	86 078	Upstream	−	T	Insertion
3	orf00256	Lactoylglutathione lyase	257 737	Upstream	−	C	Insertion
4	orf00263	Hypothetical protein	261 715	Upstream	−	T	Insertion
5	orf00263	Hypothetical protein	261 744	Upstream	−	AG	Insertion
6	orf00469	Acetaldehyde dehydrogenase	471 178	Exonic	T	C	Synonymous SNV
7	orf00501	TRAP transporter substrate-binding protein	504 022	Upstream	−	G	Insertion
8	orf00566	Phosphopantothenoylcysteine decarboxylase	568 137	Exonic	C	A	Nonsynonymous SNV
9	orf00643	ABC transporter ATP-binding protein	647 351	Upstream	A	T	Synonymous SNV
10	orf00726	TRAP transporter large permease	732 078	Exonic	−	GT	Insertion
11	orf01257	UvrY/SirA/GacA family response regulator transcription factor	1 312 231	Upstream	−	A	Insertion
12	orf02141	Branched-chain amino acid ABC transporter permease	2 212 367	Exonic	−	C	Insertion
13	orf02185	Hypothetical protein	2 262 995	Upstream	−	G	Insertion
14	orf02433	Hypothetical protein	2 529 845	Exonic	−	T	Insertion
15	orf02522	Membrane proteins	2 628 488	Exonic	−	C	Insertion
16	orf02541	Ig-like domain-containing protein	2 650 751	Exonic	G	A	Synonymous SNV
17	orf02541	Ig-like domain-containing protein	2 651 078	Exonic	A	G	Synonymous SNV
18	orf02541	Ig-like domain-containing protein	2 651 117	Exonic	G	A	Synonymous SNV
19	orf02541	Ig-like domain-containing protein	2 651 159	Exonic	C	G	Synonymous SNV
20	orf02541	Ig-like domain-containing protein	2 651 160	Exonic	G	C	Nonsynonymous SNV
21	orf02541	Ig-like domain-containing protein	2 651 163	Exonic	T	−	Deletion
22	orf02541	Ig-like domain-containing protein	2 651 165	Exonic	G	T	Nonsynonymous SNV
23	orf02541	Ig-like domain-containing protein	2 651 166	Exonic	C	G	Nonsynonymous SNV
24	orf02541	Ig-like domain-containing protein	2 651 168	Exonic	−	A	Insertion
25	orf02541	Ig-like domain-containing protein	2 651 174	Exonic	A	G	Synonymous SNV
26	orf02541	Ig-like domain-containing protein	2 651 177	Exonic	C	G	Nonsynonymous SNV
27	orf02541	Ig-like domain-containing protein	2 651 180	Exonic	C	T	Synonymous SNV
28	orf02541	Ig-like domain-containing protein	2 651 181	Exonic	G	A	Nonsynonymous SNV
29	orf02541	Ig-like domain-containing protein	2 652 098	Exonic	G	A	Synonymous SNV
30	orf02541	Ig-like domain-containing protein	2 653 040	Exonic	A	G	Synonymous SNV
31	orf02542	Hypothetical protein	2 653 448	Exonic	G	−	Deletion
32	orf02804	Hypothetical protein	2 932 441	Downstream	C	−	Deletion
33	orf02913	Hypothetical protein	3 032 494	Exonic	T	C	Synonymous SNV
34	orf03675	Urease accessory protein ureD	3 867 248	Upstream	−	G	Insertion
35	orf03864	Alcohol dehydrogenase catalytic domain-containing protein	4 050 113	Upstream	T	G	Nonsynonymous SNV

Number

ORF number

Protein name

Locus

Block

Reference

Change

Type

− means there are no base pairs at this location.

orf00034

RNA-binding protein S1

37 964

Exonic

−

Insertion

orf00080

Hypothetical protein HALTITAN3299

86 078

Upstream

−

Insertion

orf00256

Lactoylglutathione lyase

257 737

Upstream

−

Insertion

orf00263

Hypothetical protein

261 715

Upstream

−

Insertion

orf00263

Hypothetical protein

261 744

Upstream

−

Insertion

orf00469

Acetaldehyde dehydrogenase

471 178

Exonic

Synonymous SNV

orf00501

TRAP transporter substrate-binding protein

504 022

Upstream

−

Insertion

orf00566

Phosphopantothenoylcysteine decarboxylase

568 137

Exonic

Nonsynonymous SNV

orf00643

ABC transporter ATP-binding protein

647 351

Upstream

Synonymous SNV

orf00726

TRAP transporter large permease

732 078

Exonic

−

Insertion

orf01257

UvrY/SirA/GacA family response regulator transcription factor

1 312 231

Upstream

−

Insertion

orf02141

Branched-chain amino acid ABC transporter permease

2 212 367

Exonic

−

Insertion

orf02185

Hypothetical protein

2 262 995

Upstream

−

Insertion

orf02433

Hypothetical protein

2 529 845

Exonic

−

Insertion

orf02522

Membrane proteins

2 628 488

Exonic

−

Insertion

orf02541