Research Progress in the Biosynthesis and Metabolic Engineering of Hyaluronic Acid

doi:10.13560/j.cnki.biotech.bull.1985.2021-0443

Abstract

Abstract:

Hyaluronic acid（HA）,a functional glycosamine polymer widely existing in organism,presents broad potential applications in daily-use chemicals,medicine and food. With the development of synthetic biology technologies such as genetic engineering and metabolic engineering,some new challenges are emerging while the biosynthesis process and mechanism of HA have been studied in depth. This review summarizes the key genes and pathways of HA synthase from the perspective of molecular biology,aligns and analyses the amino acid sequence of HA synthase from different sources. It also describes in detail the bottleneck issues such as the safety of HA production,substrate imbalance and low oxygen solubility in fermentation,and introduces the development of strategies on corresponding synthetic biology in commonly used microbial hosts,thereby induces the effective strategies for high-yield HA by microorganism. This review may provide an important support for further promoting the green biomanufacturing of HA.

Key words: hyaluronic acid, hyaluronic acid synthase, genetic engineering, metabolic engineering, Bacillus subtilis, Corynebacterium glutamicum

MA Yan-qin, QIU Yi-bin, LI Sha, XU Hong. Research Progress in the Biosynthesis and Metabolic Engineering of Hyaluronic Acid[J]. Biotechnology Bulletin, 2022, 38(2): 252-262.

Figures/Tables 6

References 83

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Microorganism	Strategy	HA /（g·L^-1）	Molecular weight/Da	Reference
S. zooepidemicus CKD 117	Optimizing fermentation conditions	5.4	2.9×10⁶	[33]
S. zooepidemicus WSH-24	Intermittent alkaline-stress strategy	6.5	-	[5]
S. zooepidemicus ATCC 39920	Using oxygen vector and optimum impeller tip speed	4.25	1.54×10⁷	[23]
S. zooepidemicus BCRC15414	A two-stage anoxic（static）-aerobic sequential culture	0.51	3.0×10³	[22]
S. zooepidemicus ATCC 39920	Redirection of carbon flux by addition of glycolytic inhibitors,glutamine and iodoacetate	5	3.1-3.2×10⁶	[34]
S. zooepidemicus ATCC 39920	Overexpression of scrB in S. zooepidemicus ΔfruK	5.6	-	[35]
S. zooepidemicus IBRC-M 10919 Δhyl	Removal of the hyaluronidase-encoding gene	6	3.8×10⁶	[36]
S. zooepidemicus HA-13-06	Two-stage fermentation process	4.75	2.36×10⁶	[37]
S. zooepidemicus MTCC 3523	Dissolved oxygen and N-acetyl glucosamine supply	2.4	2.53×10⁶	[38]
S. thermophilus YIT 2084	Fermentation conditions and soybean peptide supplementation	0.208	1.3×10⁵	[39]
S. thermophilus YIT 2084	Co-expressing HA synthase hasA and hasB	1.2	1×10⁶	[4]
S. equisimilis mutant nc2168	Optimization of flask culture medium and conditions	0.225	-	[40]
S. iniae	Ultraviolet mutation	0.12	3×10⁵	[41]
S. sp. ID9102	Optimization of medium components	6.94	5.9×10⁶	[42]
E. coli Top10	Co-expressing HA synthase hasA and ugd from S. equisimilis	0.19	3.5×10⁵-1.9×10⁶	[43]
E. coli BL21	Expression of hyaluronan synthase hasA from S. zooepidemicus ATCC 39920	0.532	3.46×10⁴	[44]
B. subtilis 168	Inducible expression of pmHAS,tuaD and gtaB	6.8	4.55×10⁶	[45]
	Coexpression committed genes（hasA,tuaD,gtaB,glmU,glmM,and glmS）;downregulate pfkA expression of the glycolytic pathway;integration of LHAase with the activity at 1.62×10⁶ U/mL	19.38	6.62×10³	[24]
	Engineering of cell membrane by overexpressing pgsA,clsA and reducing the expression of ftsZ	1.25	2.1×10⁶	[26]
B. subtilis WB600	Regulation of temperature using the recombinant strain carrying hasA gene from S. ubris,hasB,and hasC from B. subtilis WB600	3.65	0.39-6.94×10⁶	[46]
C. glutamicum 13032	Coexpression gene hasA from S. equisimilis,hasB from C. glutamicum ATCC13032	8.3	1.3×10⁶	[30]
	Coexpression gene hasA from S. equisimilis,hasB from C. glutamicum ATCC13032;deletion of lactate dehydrogenase	21.6	1.28×10⁶	[32]
	An additional promoter PdapB for driving hasB expression;deletion of fba,zwf deletion and knockout of lactate/acetate pathway	28.7	0.21×10⁶	[47]
	Overexpression of hyaluronan synthase spHasA,enzymes of intermediate metabolic pathways and attenuation of polysaccharide biosynthesis;Disruption of the encapsulation with the leech hyaluronidase	34.2-74.1	5.3×10⁴-3.31×10⁵	[21]
Streptomyces albulus	Overexpression of gene hasA from S. zooepidemicus,gene udgA,glmU,and gtaB genes from Streptomyces avermitilis	6.2	2×10⁶	[48]
Pichia pastoris	Overexpression of xhasA2 and xhasB genes from Xenopus laevis;hasC,hasD,and hasE genes from P. pastoris	0.8-1.7	1.2-2.5×10⁶	[19]
Lactococcus lactis CES15	Expression of hasA gene under the control of the PnisA promoter	6.09	-	[49]

Microorganism	Strategy	HA /（g·L^-1）	Molecular weight/Da	Reference
S. zooepidemicus CKD 117	Optimizing fermentation conditions	5.4	2.9×10⁶	[33]
S. zooepidemicus WSH-24	Intermittent alkaline-stress strategy	6.5	-	[5]
S. zooepidemicus ATCC 39920	Using oxygen vector and optimum impeller tip speed	4.25	1.54×10⁷	[23]
S. zooepidemicus BCRC15414	A two-stage anoxic（static）-aerobic sequential culture	0.51	3.0×10³	[22]
S. zooepidemicus ATCC 39920	Redirection of carbon flux by addition of glycolytic inhibitors,glutamine and iodoacetate	5	3.1-3.2×10⁶	[34]
S. zooepidemicus ATCC 39920	Overexpression of scrB in S. zooepidemicus ΔfruK	5.6	-	[35]
S. zooepidemicus IBRC-M 10919 Δhyl	Removal of the hyaluronidase-encoding gene	6	3.8×10⁶	[36]
S. zooepidemicus HA-13-06	Two-stage fermentation process	4.75	2.36×10⁶	[37]
S. zooepidemicus MTCC 3523	Dissolved oxygen and N-acetyl glucosamine supply	2.4	2.53×10⁶	[38]
S. thermophilus YIT 2084	Fermentation conditions and soybean peptide supplementation	0.208	1.3×10⁵	[39]
S. thermophilus YIT 2084	Co-expressing HA synthase hasA and hasB	1.2	1×10⁶	[4]
S. equisimilis mutant nc2168	Optimization of flask culture medium and conditions	0.225	-	[40]
S. iniae	Ultraviolet mutation	0.12	3×10⁵	[41]
S. sp. ID9102	Optimization of medium components	6.94	5.9×10⁶	[42]
E. coli Top10	Co-expressing HA synthase hasA and ugd from S. equisimilis	0.19	3.5×10⁵-1.9×10⁶	[43]
E. coli BL21	Expression of hyaluronan synthase hasA from S. zooepidemicus ATCC 39920	0.532	3.46×10⁴	[44]
B. subtilis 168	Inducible expression of pmHAS,tuaD and gtaB	6.8	4.55×10⁶	[45]
	Coexpression committed genes（hasA,tuaD,gtaB,glmU,glmM,and glmS）;downregulate pfkA expression of the glycolytic pathway;integration of LHAase with the activity at 1.62×10⁶ U/mL	19.38	6.62×10³	[24]
	Engineering of cell membrane by overexpressing pgsA,clsA and reducing the expression of ftsZ	1.25	2.1×10⁶	[26]
B. subtilis WB600	Regulation of temperature using the recombinant strain carrying hasA gene from S. ubris,hasB,and hasC from B. subtilis WB600	3.65	0.39-6.94×10⁶	[46]
C. glutamicum 13032	Coexpression gene hasA from S. equisimilis,hasB from C. glutamicum ATCC13032	8.3	1.3×10⁶	[30]
	Coexpression gene hasA from S. equisimilis,hasB from C. glutamicum ATCC13032;deletion of lactate dehydrogenase	21.6	1.28×10⁶	[32]
	An additional promoter PdapB for driving hasB expression;deletion of fba,zwf deletion and knockout of lactate/acetate pathway	28.7	0.21×10⁶	[47]
	Overexpression of hyaluronan synthase spHasA,enzymes of intermediate metabolic pathways and attenuation of polysaccharide biosynthesis;Disruption of the encapsulation with the leech hyaluronidase	34.2-74.1	5.3×10⁴-3.31×10⁵	[21]
Streptomyces albulus	Overexpression of gene hasA from S. zooepidemicus,gene udgA,glmU,and gtaB genes from Streptomyces avermitilis	6.2	2×10⁶	[48]
Pichia pastoris	Overexpression of xhasA2 and xhasB genes from Xenopus laevis;hasC,hasD,and hasE genes from P. pastoris	0.8-1.7	1.2-2.5×10⁶	[19]
Lactococcus lactis CES15	Expression of hasA gene under the control of the PnisA promoter	6.09	-	[49]

Field	Application	HA molecular weight/Da	Reference
Medicine	Treatment of inflam- matory skin diseases.	6×10⁶	[70-72]
	Anticancer and anti- proliferative	1.2×10⁵	[73-74]
	Treatment of inflam- matory joint diseases	1×10⁶	[75-77]
	Anti-diabetic	-	[78]
	Immunomodulatory	2×10⁴	[79]
Cosmetics	Anti-aging	<1×10⁴	[80]
Cosmetics	Skin-repairing	1-10×10⁴	[66]
Food	Food and health	<1×10⁴	[81]

Field	Application	HA molecular weight/Da	Reference
Medicine	Treatment of inflam- matory skin diseases.	6×10⁶	[70-72]
	Anticancer and anti- proliferative	1.2×10⁵	[73-74]
	Treatment of inflam- matory joint diseases	1×10⁶	[75-77]
	Anti-diabetic	-	[78]
	Immunomodulatory	2×10⁴	[79]
Cosmetics	Anti-aging	<1×10⁴	[80]
Cosmetics	Skin-repairing	1-10×10⁴	[66]
Food	Food and health	<1×10⁴	[81]