Inositol Biosynthesis and Fermentation Optimization of an Inositol Biosynthesis Pathway Using Citrus sinensis Inositol Phosphate Synthase Gene Csino3

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

Abstract

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 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

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, 2026, 42(4): 345-356.

Figures/Tables 14

References 29

[1]	Kılbaş B, Balci M. Recent advances in inositol chemistry: synthesis and applications [J]. Tetrahedron, 2011, 67(13): 2355-2389.
[2]	陈庆伟. 基因工程改造大肠杆菌发酵混合糖生产肌醇的研究 [D]. 杭州: 浙江工商大学, 2023.
	Chen QW. Study on the production of inositol by fermentation of mixed sugar with genetically engineered Escherichia coli [D]. Hangzhou: Zhejiang Gongshang University, 2023.
[3]	Omoruyi FO, Stennett D, Foster S, et al. New frontiers for the use of IP6 and inositol combination in treating diabetes mellitus: a review [J]. Molecules, 2020, 25(7): 1720.
[4]	Ma AJ, Cui WX, Wang XN, et al. Osmoregulation by the myo-inositol biosynthesis pathway in turbot Scophthalmus maximus and its regulation by anabolite and c-Myc [J]. Comp Biochem Physiol Part A Mol Integr Physiol, 2020, 242: 110636.
[5]	Jiang WD, Li SG, Mi HF, et al. Myo-inositol prevents the gill rot in fish caused by Flavobacterium columnare infection [J]. Aquaculture, 2022, 546: 737393.
[6]	Shimada M, Hibino M, Takeshita A. Dietary supplementation with myo-inositol reduces hepatic triglyceride accumulation and expression of both fructolytic and lipogenic genes in rats fed a high-fructose diet [J]. Nutr Res, 2017, 47: 21-27.
[7]	杨连生, 扶雄, 孙由芳, 等. 生物酶水解植酸钙提取肌醇研究 [J]. 食品工业科技, 1997, 18(5): 48-48, 53.
	Yang LS, Fu X, Sun YF, et al. Study on drawing inositol from phytin by hydrolysis enzyme method[J]. Sci Technol Food Ind, 1997, 18(5): 48-48, 53.
[8]	王建玲, 王敏, 王春霞, 等. 利用酶法生产肌醇初步研究 [J]. 天津轻工业学院学报, 1998, 13(2): 15-19.
	Wang JL, Wang M, Wang CX, et al. Primary study on inositol production by hydrolyst[J]. J Tianjin Univ Sci Technol, 1998, 13(2): 15-19.
[9]	郭育涛, 陈坚, 伦世仪. 微生物发酵法生产肌醇的研究 [J]. 西北轻工业学院学报, 1998, 16(1): 117-120.
	Guo YT, Chen J, Lun SY. Microbiol synthesis of inositol by fermentation [J]. J Shaanxi Univ Sci Technol, 1998, 16(1): 117-120.
[10]	Li YJ, Han PP, Wang J, et al. Production of myo-inositol: Recent advance and prospective [J]. Biotechnol Appl Biochem, 2022, 69(3): 1101-1111.
[11]	Dinicola S, Unfer V, Facchinetti F, et al. Inositols: from established knowledge to novel approaches [J]. Int J Mol Sci, 2021, 22(19): 10575.
[12]	Lu YP, Wang L, Teng F, et al. Production of myo-inositol from glucose by a novel trienzymatic cascade of polyphosphate glucokinase, inositol 1-phosphate synthase and inositol monophosphatase [J]. Enzyme Microb Technol, 2018, 112: 1-5.
[13]	黄贞杰, 陈由强, 陈丽霞, 等. 代谢工程改造酿酒酵母合成肌醇 [J]. 微生物学通报, 2017, 44(10): 2289-2296.
	Huang ZJ, Chen YQ, Chen LX, et al. Metabolic engineering of Saccharomyces cerevisiae for inositol production [J]. Microbiol China, 2017, 44(10): 2289-2296.
[14]	Shiue E, Brockman IM, Prather KLJ. Improving product yields on D-glucose in Escherichia coli via knockout of pgi and zwf and feeding of supplemental carbon sources [J]. Biotechnol Bioeng, 2015, 112(3): 579-587.
[15]	Yi MH, Yang LZ, Ma J, et al. Biosynthesis of myo-inositol in Escherichia coli by engineering myo-inositol-1-phosphate pathway [J]. Biochem Eng J, 2020, 164: 107792.
[16]	朱宏宇, 王晓璐, 刘亚君, 等. 产肌醇毕赤酵母细胞工厂的优化 [J]. 微生物学通报, 2023, 50(9): 3731-3746.
	Zhu HY, Wang XL, Liu YJ, et al. Optimization of a Komagataella phaffii cell factory for producing inositol [J]. Microbiol China, 2023, 50(9): 3731-3746.
[17]	Henry AS, Gaspar LM, Jesch AS. The response to inositol: Regulation of glycerolipid metabolism and stress response signaling in yeast[J]. Chemistry and Physics of Lipids,2014,180:23-43.
[18]	邵帅, 凌宏志, 何平, 等. 自杀质粒同源重组技术敲除阴沟肠杆菌budC基因以提升乙偶姻的产率 [J]. 黑龙江大学自然科学学报, 2023, 40(4): 434-441.
	Shao S, Ling HZ, He P, et al. Increase acetoin production of Enterobacter cloacae: budC gene knockout by suicide plasmid homologous recombination technology [J]. J Nat Sci Heilongjiang Univ, 2023, 40(4): 434-441.
[19]	李莉, 张赛, 何强, 等. 响应面法在试验设计与优化中的应用 [J]. 实验室研究与探索, 2015, 34(8): 41-45.
	Li L, Zhang S, He Q, et al. Application of response surface methodology in experiment design and optimization [J]. Res Explor Lab, 2015, 34(8): 41-45.
[20]	Sarkar M, Majumdar P. Application of response surface methodology for optimization of heavy metal biosorption using surfactant modified chitosan bead [J]. Chem Eng J, 2011, 175: 376-387.
[21]	潘柯莉, 茅以诚, 岳圣杰, 等. pgi基因敲除对大肠杆菌利用混合碳源的影响 [J]. 高校化学工程学报, 2018, 32(1): 138-146.
	Pan KL, Mao YC, Yue SJ, et al. Effects of pgi knockout on mixed carbon source utilization of Escherichia coli [J]. J Chem Eng Chin Univ, 2018, 32(1): 138-146.
[22]	张芹, 王芳, 陈雅蕾, 等. 肌醇生产及应用研究进展 [J]. 中国稻米, 2012(3): 19-21.
	Zhang Q, Wang F, Chen YL, et al. Research progress of inositol production and application [J]. China Rice, 2012(3): 19-21.
[23]	Villa-García MJ, Choi MS, Hinz FI, et al. Genome-wide screen for inositol auxotrophy in Saccharomyces cerevisiae implicates lipid metabolism in stress response signaling [J]. Mol Genet Genom, 2011, 285(2): 125-149.
[24]	祁浩, 刘新利. 大肠杆菌表达系统和酵母表达系统的研究进展 [J]. 安徽农业科学, 2016, 44(17): 4-6, 52.
	Qi H, Liu XL. Research progress of expression systems of Escherichia coli and yeast [J]. J Anhui Agric Sci, 2016, 44(17): 4-6, 52.
[25]	Moon TS, Yoon SH, Lanza AM, et al. Production of glucaric acid from a synthetic pathway in recombinant Escherichia coli [J]. Appl Environ Microbiol, 2009, 75(3): 589-595.
[26]	Wu YF, Shen XL, Yuan QP, et al. Metabolic engineering strategies for co-utilization of carbon sources in microbes [J]. Bioengineering, 2016, 3(1): 10.
[27]	马健. 产肌醇的重组大肠杆菌工程菌构建及其发酵条件优化 [D]. 杭州: 浙江工商大学, 2022.
	Ma J. Construction of inositol-producing recombinant Escherichia coli and optimization of fermentation conditions [D]. Hangzhou: Zhejiang Gongshang University, 2022.
[28]	Zhang QQ, Wang XL, Luo HY, et al. Metabolic engineering of Pichia pastoris for myo-inositol production by dynamic regulation of central metabolism [J]. Microb Cell Fact, 2022, 21(1): 112.
[29]	You R, Wang L, Shi CR, et al. Efficient production of myo-inositol in Escherichia coli through metabolic engineering [J]. Microb Cell Fact, 2020, 19(1): 109.

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

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

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

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

因素 Factor	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