基于甜橙肌醇磷酸合酶基因Csino3的肌醇生物合成及发酵优化

doi:10.13560/j.cnki.biotech.bull.1985.2025-0879

摘要/Abstract

摘要：

目的原核共表达甜橙（Citrus sinensis）肌醇磷酸合酶（myo-inositol phosphate synthase）基因Csino3与大肠杆菌MG1655（Escherichia coli MG1655）肌醇-1-单磷酸酶（inositol-1-monophosphatas）基因suhB，构建高效的肌醇生物合成途径，并通过优化发酵条件和敲除大肠杆菌的pgi和gldA基因以提高肌醇产量。方法构建原核表达载体pETDuet-1-Csino3-suhB，将其转化至大肠杆菌E.coli BL21（DE3）进行异源表达。通过IPTG诱导表达，并采用SDS-PAGE鉴定可溶性蛋白表达情况。对葡萄糖浓度、IPTG浓度、接种量和初始pH值4个发酵参数进行优化，在单因素实验基础上，通过响应面法进一步优化发酵条件。随后通过敲除pgi（葡萄糖-6-磷酸异构酶）、gldA（甘油脱氢酶）改造大肠杆菌代谢途径，进一步提高肌醇产量。结果通过在E.coli BL21（DE3）中异源共表达重组质粒pETDuet-1-Csino3-suhB，证实Csino3和SuhB蛋白在25 ℃下均以可溶性形式存在。采用单因素实验和响应面分析法对目的菌株优化发酵条件，得出最佳发酵条件为：葡萄糖浓度9.7 g/L、IPTG浓度0.5 mmol/L、接种量8%、初始pH值8.1，此时肌醇发酵产量为309 mg/L。在上述发酵条件的基础上对敲除菌株ΔgldAΔpgi-pETDuet-1-Csino3-suhB/E.coli BL21（DE3）进行发酵优化，在添加20 g/L的初始甘油下，肌醇产量达到最大值3.97 g/L，比敲除前提高了12.8倍左右。结论重组菌株pETDuet-1-Csino3-suhB /E.coli BL21（DE3）能够有效产生肌醇，通过敲除pgi、gldA基因改变大肠杆菌代谢通路能极大提高肌醇产量。

关键词: 甜橙, 肌醇合成途径, 原核共表达, 基因敲除, 发酵条件优化, 响应面实验

Abstract:

Objective To establish an efficient myo-inositol biosynthetic pathway through prokaryotic co-expression of Citrus sinensis myo-inositol phosphate synthase gene Csino3 and Escherichia coli MG1655 inositol-1-monophosphatase gene suhB. To increase the inositol yield by optimizing fermentation conditions and knocking out pgi and gldA genes in E. coli. Method The prokaryotic co-expression vector pETDuet-1-Csino3-suhB was constructed and transformed into E. coli BL21(DE3) for heterologous expression. Protein expression was induced with IPTG and the expression of soluble protein was identified via SDS-PAGE analysis. Four fermenting parameters, glucose concentration, IPTG concentration, inoculum size, and initial pH were optimized through single-factor experiments. Further fermentation condition was optimized using response surface methodology. Then Enhanced myo-inositol yield was enhanced by knocking out the pgi gene (encoding glucose-6-phosphate isomerase) and the gene gldA (glycerol dehydrogenase) to modify the metabolic pathway of E. coli . Result Successful heterologous co-expression of the recombinant plasmid pETDuet-1-Csino3-suhB in E. coli BL21(DE3) confirmed that both Csino3 and SuhB proteins were expressed in soluble forms under 25 ℃ induction. The fermentation conditions for the target strain were optimized using single-factor experiments and response surface methodology (RSM). The optimal conditions were determined as follows: glucose concentration 9.7 g/L, IPTG concentration 0.5 mmol/L, inoculum size 8%, and initial pH 8.1. Under these conditions, myo-inositol production reached 309 mg/L. Based on the above mentioned fermentation conditions, further optimization was performed for the knockout strain △gldAΔpgi-pETDuet-1-CSino3-suhB/E.coli BL21(DE3). Under an initial glycerol concentration of 20 g/L, the myo-inositol production reached 3.97 g/L, representing an approximately 12.8-fold increase compared to the pre-knockout strain. Conclusion The recombinant strain pETDuet-1-CSino3-suhB /E.coli BL21(DE3) can efficiently produce inositol. Knocking out the pgi and gldA gene to redirect the metabolic pathway of E. coli significantly enhances inositol yield.

Key words: Citrus sinensis, inositol biosynthesis pathway, prokaryotic co-expression, gene knockout, optimization of fermentation condition, response surface experiment

颜鑫, 吴筱, 何思宇, 段玉欢, 仇伍霞, 袁晓琴, 毛新芳, 刘忠渊. 基于甜橙肌醇磷酸合酶基因Csino3的肌醇生物合成及发酵优化[J]. 生物技术通报, doi: 10.13560/j.cnki.biotech.bull.1985.2025-0879.

YAN Xin, WU Xiao, HE Si-yu, DUAN Yu-huan, QIU Wu-xia, YUAN Xiao-qin, MAO Xin-fang, LIU Zhong-yuan. Inositol Biosynthesis and Fermentation Optimization of an Inositol Biosynthesis Pathway Using Citrus sinensis Inositol Phosphate Synthase Gene Csino3[J]. Biotechnology Bulletin, doi: 10.13560/j.cnki.biotech.bull.1985.2025-0879.

图/表 14

表1 敲除基因pgi、gldA所用引物序列

Table 1 Primer sequences for knockout of genes pgi and gldA

引物名称 Primer name	引物序列 Primer sequence （5′-3′）
pgi-outF	CCACTGTGCCTGAACATCGCGTTCGC
pgi-outR	GCAGGTCGCTTCACGACTCTTCTCCAGAGG
pgi-inF	GCTGCACGTAGCGCTGCGTAACCG
pgi-inR	GCACTTCCGCGATGTGAGTCCCATCGAC
gldA-outF	CCCCACCATCCGCCAGTTAAACAGCA
gldA-outR	CTGGACACCGCTAACGTCGCAGAAGTC
gldA-inF	GGAGTCACTACATGCTGTTCGGCAG
gldA-inR	CTACACCGATGAGGGTGAGTTTGACC

表2 pETDuet-1- Csino3-suhB质粒克隆位点

Table 2 Cloning sites of pETDuet-1-Csino3-suhB plasmid

引物名称 Primer name	引物序列 Primer sequence （5′-3′）	限制性酶切位点 Restriction enzyme site
CsIno3上游	GGATCCATGTTTATCGAAAATTTCAAGGTT	BamH I
CsIno3下游	TTGTACTAAAACCTTATGTTCACTAAGCTT	Hind III
suhB 上游	CATATGATGCATCCGATGCTGAACATCGCC	Nde I
suhB 下游	GAGTTAAGCGACGCTCTGAAGCGTCTCGAG	Xho I

图1 重组pETDuet-1-Csino3-suhB/BL21（DE3）在25 ℃的诱导表达M：蛋白质分子量标准；1：IPTG诱导前细菌裂解液；2：IPTG诱导后的细菌裂解液；3：超声破碎后上清液；4：超声破碎后沉淀

Fig. 1 Induced expression of recombinant pETDuet-1-Csino3-suhB/BL21(DE3) at 25 ℃M: Protein molecular weight marker. 1: Lysis of bacteria without induction by IPTG. 2: Lysis of bacteria after induction by IPTG. 3: Supernatant after ultrasonic crushing. 4: Sedimentation after ultrasonic crushing

图2 Csino3蛋白纯化M：蛋白质分子量标准；1：流穿液；2-6：分别为10 、50、100 、200 、400 mmol/L咪唑洗脱液

Fig. 2 Csino3 protein purificationM: Protein molecular weight standard. 1: Flow-through; 2-6: 10, 50, 100, 200, 400 mmol/L imidazole elution, respectively

图3 肌醇标准品超高相液相质谱图

Fig. 3 Ultra-high-phase liquid mass spectrometry of myo-inositol

图4 样品超高相液相质谱图

Fig. 4 Ultra-high-phase liquid mass spectrometry of samples

图5 单因素发酵工艺优化A：IPTG终浓度；B：葡萄糖浓度；C：接种量；D：初始pH；E：时间

Fig 5 Optimization of single-factor fermentationA: Final concentration of IPTG. B: Glucose concentration. C: Inoculation amount. D: Initial pH. E: Time

表3 Box-Behnken design设计因素及水平表

Table 3 Factor and level table of Box-Behnken design

因素 Factor	单位 Unit	Level	Low level	High level
Glucose	g/L	10.0	5.0	15.0
IPTG	mmol/L	0.5	0.2	0.8
Inoculation amount	%	8.0	6.0	10.0
Initial pH	-	8.0	7.0	9.0

表4 Box-Behnken 响应面试验设计及结果

Table 4 Box-Behnken response surface experimental design and results

Number	A （g/L）	B （mmol/L）	C （%）	D	Inositol （mg/L）
1	5	0.2	8	8	151.00
2	15	0.2	8	8	117.33
3	5	0.8	8	8	159.67
4	15	0.8	8	8	140.33
5	10	0.5	6	7	149.33
6	10	0.5	10	7	146.00
7	10	0.5	6	9	156.00
8	10	0.5	10	9	178.33
9	5	0.5	8	7	121.00
10	15	0.5	8	7	114.33
11	5	0.5	8	9	152.67
12	15	0.5	8	9	143.00
13	10	0.2	6	8	152.33
14	10	0.8	6	8	166.67
15	10	0.2	10	8	145.33
16	10	0.8	10	8	144.33
17	5	0.5	6	8	144.67
18	15	0.5	6	8	138.00
19	5	0.5	10	8	176.67
20	15	0.5	10	8	137.33
21	10	0.2	8	7	174.33
22	10	0.8	8	7	153.00
23	10	0.2	8	9	191.67
24	10	0.8	8	9	177.00
25	10	0.5	8	8	288.33
26	10	0.5	8	8	272.33
27	10	0.5	8	8	287.00
28	10	0.5	8	8	295.67
29	10	0.5	8	8	298.00

图6 响应面优化菌株pETDuet-1-Csino3-suhB/BL21（DE3）生产肌醇A：IPTG和葡萄糖；B：接种量和葡萄糖；C：pH和葡萄糖；D：接种量和IPTG；E：pH和IPTG；F：pH和接种量

Fig. 6 Response surface on inositol production optimized by strain pETDuet-1-Csino3-suhB/BL21(DE3)A: IPTG and glucose. B: Inoculation amount and glucose. C: pH and glucose. D: Inoculation amount and IPTG. E: pH and IPTG. F: pH and inoculation amount

表5 Box-Behnken 响应面试验方差分析

Table 5 Analysis of variance (ANOVA) for Box-Behnken response surface methodology

变异来源 Source of variation	平方和 SS	自由度 df	均方 MS	F值 F-value	P值 P-value	备注 Note
Model	83 481.48	14	5 962.96	25.45	<0.000 1	**
A- Glucose	1 108.48	1	1 108.48	4.73	0.047 2	*
B- IPTG	6.75	1	6.75	0.028 8	0.867 6	Not significant
C- Inoculation amount	36.75	1	36.75	0.156 9	0.698 0	Not significant
D- Initial pH	1 648.93	1	1 648.93	7.04	0.018 9	**
AB	51.36	1	51.36	0.219 2	0.646 8	Not significant
AC	266.78	1	266.78	1.14	0.304 0	Not significant
AD	2.25	1	2.25	0.009 6	0.923 3	Not significant
BC	58.78	1	58.78	0.250 9	0.624 2	Not significant
BD	11.11	1	11.11	0.047 4	0.830 7	Not significant
CD	164.69	1	164.69	0.703 0	0.415 9	Not significant
A²	45 114.14	1	45 114.14	192.57	<0.000 1	**
B²	24 352.12	1	24 352.12	103.94	<0.000 1	**
C²	28 274.17	1	28 274.17	120.69	<0.000 1	**
D²	26 002.04	1	26 002.04	110.99	<0.000 1	**
Residual	3 279.91	14	234.28
Lack of fit	2 874.94	10	287.49	2.84	0.1632	Not significant
Pure error	404.98	4	101.24
Cor total	86 761.39	28

图7 BL21（DE3）/Δpgi菌落PCR验证M：DNA Marker；A-1：无模板阴性对照扩增结果；A-2：原始菌株的内部引物扩增结果；A-（3-16）：第1-14号克隆的内部引物扩增结果；B-1：无模板阴性对照的扩增结果；B-2：阴性克隆外侧引物的扩增结果

Fig. 7 Colony PCR verification of BL21(DE3)/ΔpgiM: DNA Marker. A-1: No-template negative control amplification result. A-2: Internal primer amplification result of the original strain. A-(3-16): Internal primer amplification results of clone 1-14. B-1: No-template negative control amplification result. B-2: External primer amplification result of negative clone

图8 BL21（DE3）/△gldA 菌落PCR验证M：DNA Marker；A-1：无模板阴性对照扩增结果；A-2：原始菌株的内部引物扩增结果；A-（3-13）：第1-11号克隆的内部引物扩增结果；B-1：阴性克隆外侧引物的扩增结果；B-2：无模板阴性对照的扩增结果

Fig. 8 Colony PCR verification of BL21(DE3)/△gldAM: DNA Marker. A-1: No-template negative control amplification result. A-2: Internal primer amplification result of the original strain. A-(3-13): Internal primer amplification results of clone 1-11. B-1: External primer amplification result of negative clone. B-2: No-template negative control amplification result

图9 敲除pgi、gldA基因菌株合成肌醇能力A：3种菌株生产肌醇含量；B：不同甘油浓度对△pgi-pETDuet-1-Csino3-suhB / BL21（DE3）发酵肌醇的影响；C：不同甘油浓度对△gldA△pgi -pETDuet-1-Csino3-suhB / BL21（DE3）发酵肌醇的影响

Fig. 9 Inositol-synthezing capacity of the pgi- and gldA-knockout strainA: Inositol production content by three strains. B: Effect of different glycerol concentrations on inositol fermentation by △pgi-pETDuet-1-Csino3-suhB/BL21(DE3). C: Effect of different glycerol concentrations on inositol fermentation by △gldA△pgi-pETDuet-1-Csino3-suhB/BL21(DE3)

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