Eliminating the capsule-like layer to promote glucose uptake for hyaluronan production by engineered corynebacterium glutamicum

ABSTRACT Hyaluronan is widely used in cosmetics and pharmaceutics. Development of robust and safe cell factories and cultivation approaches to efficiently produce hyaluronan is of many

interests. Here, we describe the metabolic engineering of _Corynebacterium glutamicum_ and application of a fermentation strategy to manufacture hyaluronan with different molecular weights.

_C. glutamicum_ is engineered by combinatorial overexpression of type I hyaluronan synthase, enzymes of intermediate metabolic pathways and attenuation of extracellular polysaccharide

biosynthesis. The engineered strain produces 34.2 g L−1 hyaluronan in fed-batch cultures. We find secreted hyaluronan encapsulates _C. glutamicum_, changes its cell morphology and inhibits

metabolism. Disruption of the encapsulation with leech hyaluronidase restores metabolism and leads to hyper hyaluronan productions of 74.1 g L−1. Meanwhile, the molecular weight of

hyaluronan is also highly tunable. These results demonstrate combinatorial optimization of cell factories and the extracellular environment is efficacious and likely applicable for the

production of other biopolymers. SIMILAR CONTENT BEING VIEWED BY OTHERS REGULATING CELLULAR METABOLISM AND MORPHOLOGY TO ACHIEVE HIGH-YIELD SYNTHESIS OF HYALURONAN WITH CONTROLLABLE

MOLECULAR WEIGHTS Article Open access 28 February 2025 PRODUCTION AND PURIFICATION OF HIGHER MOLECULAR WEIGHT CHONDROITIN BY METABOLICALLY ENGINEERED _ESCHERICHIA COLI_ K4 STRAINS Article

Open access 06 August 2020 STRUCTURAL INSIGHTS INTO TRANSLOCATION AND TAILORED SYNTHESIS OF HYALURONAN Article Open access 25 September 2024 INTRODUCTION Hyaluronan (hyaluronic acid or HA)

is a negatively charged, non-sulfated glycosaminoglycan comprising repeating uridine diphosphate glucuronate (UDP-GlcA) and uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) disaccharide

units. It is a natural substance in vertebrates and mainly found in the eyes, joints and skin, where it absorbs large amounts of water for joint lubrication1, cell coating or repair of

damaged skin tissue2. In addition to vertebrates, some pathogenic microorganisms, such as group A _Streptococcus_3 and _Pasteurella multocida_ type A strains4, produce HA as the major

component of their capsules for protection against exterior damage. Due to its biocompatibility, hygroscopicity and non-immunogenicity, HA and its derivatives are important to the cosmetics,

pharmaceutical, biomedical, and food industries5,6,7,8. Currently, commercial production of HA is mainly dependent on fermentation of group A _Streptococcus_9; however, high risk of

pathogenicity to livestock and contamination by exotoxins in the HA product hinder its broader application. The development of metabolic engineering allows the engineering of non-pathogenic

_Escherichia coli_ strains for heterologous HA production10,11. To further eliminate potential safety concerns, many generally recognized as safe (GRAS) strains, such as _Bacillus

subtilis_12,13,14,15,16, _Lactococcus lactis_17,18,19,20, _Corynebacterium glutamicum_21,22, and _Pichia pastoris_23, have been engineered as alternative producers of HA. Additionally,

cell-free systems have been exploited to produce HA at specific molecular weight (MW)24,25. Various strategies have been adopted to construct these HA producers, with selection of HA

synthases and host strains representing the most critical step, as variations in HA synthase sequence, structural conformation26, or host cell metabolic capacities make differences in HA

yield or MW. HA synthase activities have been improved by protein engineering24,27 or modification of the microenvironment of the enzymatic reaction, including the membrane lipid

composition28. Moreover, metabolic engineering strategies such as overexpressing enzymes of intermediate metabolic pathways (e.g., UDP-glucose 6-dehydrogenase or glucosamine-1-phosphate

N-acetyltransferase) or blocking the synthesis of unwanted metabolites (e.g., l-lactate) were adopted to drive the generation of intermediate metabolites required for HA synthesis29,30. In

view of the GRAS status and metabolic capacity, _C. glutamicum_ as the Gram-positive model organism has been engineered as an HA producer. In 2014, Hoffmann et al.21 first constructed the HA

biosynthetic pathway by expressing _Streptococcus equi_ subsp. _zooepidemicus_ HA synthase (seHasA) in _C. glutamicum_. The engineered strain produced 1.2 g L−1 of HA. In 2016, Cheng et

al.31 co-expressed the codon-optimized _Streptococcus dysgalactiae_ subsp. _equisimilis ssehasA_ gene along with endogenous _ugdA_ in _C. glutamicum_ to produce 8.3 g L−1 HA in fed-batch

cultivation. Additionally, deletion of _ldh_, encoding lactate dehydrogenase, increased HA production to 21.6 g L−1 in fed-batch culture29. A follow-up study enhanced the HA yield to 28.7 g 

L−1 by attenuating the glycolysis pathway, pentose phosphate pathway and the dehydrogenation of pyruvate22. In the present study, we engineer _C. glutamicum_ for high-yield HA production by

selecting the most productive HA synthase, overexpressing enzymes of the intermediate pathways to convert glucose into the HA building blocks UDP-GlcA and UDP-GlcNAc and decreasing

endogenous extracellular polysaccharide biosynthesis. The engineered strain produces 34.2 g L−1 HA in fed-batch cultures. Our analysis of cell morphology reveals that the secreted HA forms

an HA capsule-like layer, which is subsequently found to restrict nutrient uptake and inhibit HA synthesis. To relieve this inhibition effect, we supplement leech hyaluronidase (LHYal,

hydrolase)32 to the fed-batch culture to disrupt cell encapsulation and decrease broth viscosity. This strategy significantly promotes glucose uptake and HA production. RESULTS CONSTRUCTION

OF HA-PRODUCING _C. GLUTAMICUM_ STRAINS HA synthase polymerizes HA chains using two building blocks: UDP-GlcA and UDP-GlcNAc (Fig. 1a). Two types of bacterial HA synthases have been

identified: type I synthase is a bifunctional enzyme that polymerizes the HA chain, as well as transfers HA across the cell membrane33,34,35 (Supplementary Fig. 1); and type II HA synthase,

found only in _P. multocida_36, is a cytosolic glycosyltransferase with high affinity to the cytoplasmic membrane (Supplementary Fig. 1). Here, HA synthases from _P. multocida_ (pmHasA), _S.

pyogenes_ (spHasA), _S. uberis_ (suHasA), and_ S. equi_ subsp. _zooepidemicus_ HasA (seHasA) were selected and overexpressed individually in the host strain _C. glutamicum_ ATCC 13032.

Comparatively, spHasA generated the highest HA titer (1.5 g L−1) in shake-flask cultivation, which was 10-fold and 7-fold higher than that of seHasA and pmHasA, respectively (Fig. 1b).

Almost no HA was synthesized by suHasA (Fig. 1b). Protein alignment showed seHasA21 and sseHasA22,29,31 share the same protein sequence. In comparison, there is 72% sequence identity between

seHasA and spHasA and 71% sequence identity between suHasA and spHasA (Supplementary Fig. 2a). Western Blot demonstrated suHasA expressed very poorly in _C. glutamicum_ while the expression

levels of spHasA and seHasA are similar (Supplementary Fig. 2b). To further explore the reason of the distinct HA synthesis capabilities between seHasA and spHasA, we replaced the most

dissimilar region of seHasA from spHasA, the first transmembrane helix Leu7-Val25 of seHasA with the corresponding transmembrane helix Thr7-Met25 of spHasA35 to generate seHasAThr7-Met25.

The mutant seHasAThr7-Met25 was expressed to the same level of the wild-type seHasA in _C. glutamicum_ (Supplementary Fig. 2b). However, seHasAThr7-Met25 produced much higher amount of HA

(0.87 g L−1, Supplementary Fig. 2c), suggesting the first tranmembrane helix of type I HA synthase should play critical roles in regulating HA synthesis. After determining spHasA as the best

performing HA synthase, all genes encoding enzymes related to intermediate metabolite-synthesis pathways, including _galU_, _ugdA_, _glmS_, _glmM_, and _glmU_, from five species (_C.

glutamicum_ ATCC13032, cg; _B. subtilis_ 168, bs; _S. equi_ subsp. _zooepidemicus _WSH-24, se; _Pseudomonas putida_ KT2440, pt; and _E. coli_ MG1655, ec) were selected and individually

overexpressed to examine their contributions to HA formation. In particular, _ugdA_ genes (also known as _kfiD_) found in the heparosan-biosynthesis genome island of _E. coli_ O10:K5:H4 ATCC

23506 (eco) and _E. coli_ Nissle 1917 (ecn) (Supplementary Fig. 3) were investigated. We found that UDP-glucose 6-dehydrogenase (UgdA) from different species exhibited different abilities

to promote HA biosynthesis (4.5 g L−1, Fig. 1c). Endogenous UgdA encoded by _cgugdA2_ in _C. glutamicum_ showed the best performance in enhancing HA biosynthesis as compared with GalU from 

_S. equi_ subsp. _zooepidemicus_, GlmS, GlmM from _P. putida_ and GlmU from _B. subtilis_, which showed similar capabilities at stimulating HA biosynthesis (around 2 g L−1) (Fig. 1d and

Supplementary Fig. 4a–c). Therefore, we overexpressed _cgugdA2_ along with_ S. equi_ subsp. _zooepidemicus_ _ galU2_ (_segalU2_), _P. putida glmS_ (_ptglmS_) and _glmM_ (_ptglmM_) and _B.

subtilis glmU_ (_bsglmU_), respectively, finding that co-overexpression of _cgugdA2_ with _ptglmS_ or _ptglmM_ improved HA production to 5.4 g L−1 and 5.0 g L−1, respectively, in shake-flask

cultivations (Fig. 1e). By contrast, other combinations failed to further improve the HA synthesis (Fig. 1e). ENHANCEMENT OF HA PRODUCTION BY ATTENUATING THE BIOSYNTHESIS OF EXTRACELLULAR

POLYSACCHARIDES We then investigated a pathway competing with HA synthesis, specifically the biosynthesis of extracellular polysaccharides, which is involved in consumption of key

intermediate metabolites such as UDP-glucose37, the precursor of UDP-GlcA (Fig. 1a). The corynebacterial cell wall contains a peptidoglycan-attached arabinogalactan structure, which is

enveloped by mycolic acid and an outer layer of extracellular polysaccharides, proteins and lipids38 (Fig. 2a). The main components of corynebacterial extracellular polysaccharides (also

considered as capsular polysaccharides or cell-surface polysaccharides) are arabinomannan, mannan and glucan38,39. However, the genes required for biosynthesis of _C. glutamicum_

cell-surface polysaccharides remain largely unknown. Taniguchi et al.40 reported that overexpression of _C. glutamicum_ sigma factor SigD induced secretion of carbohydrate compounds and

raised the expression levels of three glycosyltransferases genes (_cg0420_, _cg0532_, and _cg1181_). Following genome analysis, we found that _cg0420_ locates in close proximity to the

_wzx_, _wzy_, and _wzz_ genes (Fig. 2b). Because the Wzx/Wzy-dependent assembly pathway synthesizes the majority of bacterial cell-surface polysaccharides (Fig. 2a)41, we speculated that

_cg0420_ may participate in biosynthesis of _C. glutamicum_ extracellular polysaccharides. Prior to _cg0420_ deletion, we found three other putative glycosyltransferase genes within the same

genomic region (_cg0424_, _cg0419_, and _cg0438_) (Fig. 2b and Supplementary Fig. 5). After many rounds of trials, we did not obtain single deletions for _cg0420_, _cg0419_, or _cg0438_.

However, we were able to delete _cg0424_ to generate strain Delcg0424. Basing on Delcg0424, we further deleted _cg0420_ and constructed strain Delcg0420,0424. It was found that neither

single _cg0424_ deletion nor double deletion of _cg0420_ and _cg0424_ impaired cell growth (Fig. 2c). Sugar constituent analysis of the Delcg0424 extracellular polysaccharides showed that

the concentration of mannose decreased by 32% while the concentration of arabinose was not affected (Fig. 2d). The concentrations of mannose and arabinose released from strain Delcg0420,0424

extracellular polysaccharides hydrolysis decreased by 24% and 47% respectively, comparing to that of the strain Delcg0424 (Fig. 2d). Thus, Cg0424 and Cg0420 probably have the activities of

mannosyltransferase and arabinosyltransferase to catalyze the formation of mannan and arabinomannan, respectively. Moreover, the synthesis of _C. gultamicum_ cell-surface polysaccharides

probably involves more glycosyltransferases, as the double deletion of _cg0420_ and _cg0424_ does not abolish the synthesis of mannan or arabinomannan. Deletion of these putative

glycosyltransferase genes positively influenced HA synthesis, as HA yield increased by 14.9% with double deletion of _cg0424_ and _cg0420_ to 6.4 g L−1 in shake-flask cultures (Table 1). The

optimal engineered _C. glutamicum_ strain CgspH-7 underwent fed-batch cultivation. After consuming 166 g L−1 glucose, the HA titer reached 34.2 g L−1 (Fig. 2e) and the broth turned viscous

(Supplementary Table 1). HA ACCUMULATION ENCAPSULATES _C. GLUTAMICUM_ CELLS HA is produced as the main component of some bacterial capsules and is attached to the cell surface to provide

protection and virulence. To determine how HA is released into culture broth and its distribution on the _C. glutamicum_ surface, we visualized the HA-producing strains CgspH-2 and CgspH-7

by phase-contrast microscopy and found that both strains exhibited cell aggregation along with HA accumulation (Fig. 3a). Moreover, the engineered _C. glutamicum_ strains showed a chain-like

morphology (Fig. 3a and Supplementary Fig. 6) resembling that of the native HA-producing _Streptococcus_ species. To visualize HA distribution, we performed negative staining with nigrosin,

a classical capsule-staining dye42. Secreted HA formed a capsule-like layer, with HA-producing cells encapsulated (Fig. 3a), whereas no similar structure was observed in the control strain

_C. glutamicum_ pXMJ-pEC carrying blank vectors. As a consequence, we proposed a model of the encapsulation process (Fig. 3b). HA molecules were not secreted directly into the medium but

rather knitted into a slime layer encapsulating the dividing cells. Additionally, the HA molecules are gradually released from the outer layer and dissolved in the culture broth. In the

presence of adequate glucose, the size of the cell capsule grow continuously (Fig. 3a between time points 16 and 28 hour), whereas decreased glucose availability at the late growth phase

made HA secretion slow down and HA encapsulation decay (Fig. 3a, time point 32 hour). THE HA CAPSULE LAYER RESTRICTS NUTRIENT UPTAKE The HA capsule layer of _Streptococcus_ plays important

roles in resisting phagocytosis and protecting the cells from desiccation and drying3. However, the influence of the HA capsule layer on cell metabolism remains unclear. Therefore, we used

recombinant LHYal with high substrate specificity to disrupt the HA capsule layer of the native HA producer _S. equi_ subsp. _zooepidemicus_ WSH-24 to study the impact of HA capsule on cell

metabolism9. Supplementation with LHYal (6000 U mL−1) disrupted the HA capsule of _S. equi_ subsp. _zooepidemicus_ (Fig. 4a). Additionally, the disruption of capsule sped up glucose

consumption, stimulated cell growth and significantly increased HA yield (Fig. 4b). This result demonstrates that the presence of an HA capsule inhibited nutrient uptake and cell growth. The

influence could be relieved by disruption of cell encapsulation by LHYal treatment (Fig. 4c). Moreover, we supplied different concentrations of high-MW HA (1.3 MDa) to cultures of _C.

glutamicum_ ATCC 13032, a non-HA producer, to examine the impact of HA that released into culture broth on cell growth and metabolism. In order to represent better the natural phenomenon,

the HA supplemented here is produced by the natural HA producer _S. equi_ subsp. _zooepidemicus_. We found significant decreases in cell growth and glucose consumption in the presence of 5 g

 L−1 HA and 10 g L−1 HA (Fig. 4d). By contrast, supplementing equal concentrations of HA pretreated with 10000 U mL−1 of LHYal for 8 hours did not slow down cell growth or glucose

consumption (Fig. 4d). These results suggest that increased concentration of high-MW HA released into cultures also restricts nutrient uptake. ELIMINATING THE CAPSULE-LIKE LAYER PROMOTES HA

PRODUCTION We then determined whether elimination of the capsule-like layer of CgspH-7 would also be beneficial to cell growth and HA production. We supplemented 6000 U mL−1 LHYal to CgspH-7

shake-flask cultures upon initiation of HA accumulation. As expected, cell encapsulation did not show up when LHYal was supplemented and the chain-structured cell aggregates partially

recovered to the natural rode shape of _C. glutamicum_ (Fig. 5a). As a result, the glucose consumption and cell growth of CgspH-7 were enhanced and HA synthesis was accordingly promoted. No

such effects were observed on the control strain pXMJ-pEC (Fig. 5b). Furthermore, addition of LHYal to fed-batch cultures of _C. glutamicum_ CgspH-7 not only improved HA production, but also

resulted in stronger metabolic activities and prolonged exponential phases (Fig. 6a–c). Supplementation of LHYal to a final concentration of 1500, 3000, or 6000 U mL−1 increased the total

consumed glucose from 166 g L−1 (Fig. 2e) to 316 g L−1 (Fig. 6a), 424 g L−1 (Fig. 6b), and 440 g L−1 (Fig. 6c), respectively and increases in the OD600 from 172 (Fig. 2e) to 215, 253, and

231 (Fig. 6a–c), respectively. Additionally, this improved HA production to 46.2 g L−1 (Fig. 6a), 57.5 g L−1 (Fig. 6b), and 74.1 g L−1 (Fig. 6c), whereas the weight average MW of HA

decreased to 155 kDa, 91 kDa and 53 kDa, respectively (Table 2). These results demonstrated that elimination of the capsule-like layer recovered cell metabolism rate and improved HA

production. DISCUSSION Construction of biosynthesis pathways of HA and other glycosaminoglycans in genetically engineered microbes represents a green and safe method for production of these

invaluable compounds. To construct an efficient HA-producing cell factory, selection of a proper HA synthase is critical, given that HA productivity and MW are largely determined by synthase

activity19. Most HA synthases are found in vertebrates, whereas bacterial HA synthases are mainly found in the group A _Streptococcus_3. Streptococcal HasA has four transmembrane helices

and two membrane-associated helices33,34,35; therefore, it is conceivable that the membrane microenvironment influences HasA activity. In the present study, we found that spHasA displayed 

stronger HA-synthesis activities in _C. glutamicum_ and resulted in higher HA accumulation relative to HasA from the other species (Fig. 1b). The differences in the sequences of these

enzymes were primarily found in the transmembrane region (Supplementary Fig. 2a), suggesting that spHasA might be better suited to the _C. glutamicum_ cytoplasmic membrane. Recently,

Westbrook et al.27 successfully enhanced the activity of _Streptococcus equisimilis_ HasA (sseHasA) in _B. subtilis_ by regulating membrane cardiolipin content and distribution.

Additionally, previous studies engineered _E. coli_ species as alternative HA producers10,11,43. However, _E. coli_ exhibit a low degree of activity by heterologously expressed HA synthases,

possibly because of the inherent incompatibility between HA synthase and the Gram-negative _E. coli_ cell structure: _E. coli_ cells have two membranes, whereas the type I HA synthase from

Gram-positive _Streptococcus_ secretes HA chains only through the inner membrane. Protein engineering of HasA44, especially changes in the transmembrane region, might represent a possible

direction for further improvements in HA production (Supplementary Fig. 2b, c). In addition to overexpressing UgdA and GlmS for precursor generation (Fig. 1a), deletion of the competing

pathway to increase the supply of UDP-glucuronate and UDP-GlcNAc is a commonly used strategy22,29. In _C. glutamicum_, there is less availability of UDP-GlcA relative to UDP-GlcNAc, given

that overexpression of UgdA improved HA yield by 2-fold (Fig. 1c). Additionally, UDP-GlcNAc is naturally produced for peptidoglycan biosynthesis (Fig. 1a), which is critical for cell

survival. UDP-glucose, the precursor of UDP-GlcA, is used to construct the outmost layer of the _C. glutamicum_ cell wall38, which should be less important to the cell than peptidoglycans

for maintaining the cell-envelope structure. A previous report showed that _cg0420_ expression enhanced the biosynthesis of extracellular polysaccharides40. In the present study, we selected

four putative genes encoding glycosyltransferases (_cg0420_, _c__g0424_, _cg0419_, and _cg0438_) and located in close proximity to _galU_ and _ugdA_ for deletion, finding that loss of

_cg0424_ and _cg0420_ reduced cell-surface polysaccharides such as mannan and arabinomannan (Fig. 2d) and enhanced HA production (Table 1) without impairing cell growth (Fig. 2c). These

results demonstrated that the enzymes encoded by _cg0420_40 and _cg0424_ are involved in extracellular polysaccharide synthesis and that the outmost layer of the _C. glutamicum_ cell wall38

comprising extracellular polysaccharides is likely not essential to cell growth. The feedback effects of HA accumulation on cell morphology and metabolism have previously been ignored. Here,

we found that during the later period of fermentation, cells of the engineered _C. glutamicum_ strain were encapsulated following HA accumulation. The formation of a capsule-like layer

(Fig. 3a) inhibited cell metabolism and growth (Fig. 4b). The morphology of the _C. glutamicum_ CgspH-7 cells resembled the chain shape of native HA-producing _Streptococcus_ species (Fig. 

4a). Moreover, our results suggest that the capsule of pathogens not only protects cells against various stressors or phages or acts as a virulence factor, but also could adversely affect

cell metabolism (Fig. 4b). This could explain why capsule formation occurs under so complex regulatory processes45. In conclusion, optimization of the HA-biosynthesis pathway, inactivation

of extracellular polysaccharide biosynthesis, destruction of cell encapsulation and resolving the mass-transfer bottleneck by supplementation with LHYal resulted in an efficient _C.

glutamicum_ cell factory and a fermentation approach. This engineered strain allowed generation of an average of 74.1 g L−1 HA with an MW of 53 kDa in 5-L fed-batch cultures (Fig. 6c and

Table 2). The results and strategies reported here are likely applicable to the production of other biopolymers. METHODS PLASMIDS AND STRAINS Plasmids and strains used in this study are

listed in Supplementary Data 1 and Supplementary Data 2. We used _E. coli_ JM109 for plasmid amplification and recombinant plasmid construction. _C. glutamicum_ ATCC 13032 was used as the

parental strain for breeding all engineered _C. glutamicum_ strains. HA synthase genes (_hasA_) were amplified from the genome of _S. equi_ subsp. _zooepidemicus_ (se) or synthesized by

GENEWIZ (Suzhou, China) according to the published genome sequences of _Streptococcus pyogenes_ (sp), _Streptococcus uberis_ (su) and _Pasteurella multocida_ (pm). The _hasA_ genes were

prefixed with “se”, “sp”, “su”, and “pm” in order to indicate species origins. After amplification with the designated primers sehasA-F and sehasA-R, sphasA-F and sphasA-R, suhasA-F and

suhasA-R and pmhasA-F and pmhasA-R (Supplementary Data 3), _hasA_ genes were ligated into HindIII/BamHI linearized pXMJ19 using the T5 exonuclease DNA assembly (TDEA) method46 to generate

the plasmids pXMJ19-sehasA, pXMJ19-sphasA, pXMJ19-suhasA, and pXMJ19-pmhasA. With the  former three  plasmids as templates, the linear form of plasmids pXMJ19-sphasA-6His,

pXMJ19-sehasA-6His, pXMJ19-suhasA-6His, pXMJ19-sehasAThr7-Met25 were generated via PCR using designated primers listed in Supplementary Data 3. The PCR products carrying homogenous 5′ and 3′

terminals (included in the designed primers) were transformed into _E. coli_ JM109 after purification and cyclized by the endogenous DNA recombinases. The plasmid

pXMJ19-sehasAThr7-Met25-6His was constructed by PCR in the same way using pXMJ19-sehasAThr7-Met25 as template. Genes encoding enzymes capable of synthesizing HA building blocks, including

_galU_ (encoding glucose-1-phosphate uridylyltransferase), _ugdA_ (encoding UDP-glucose 6-dehydrogenase), _glmM_ (encoding phosphoglucosamine mutase), _glmS_ (encoding

l-glutamine:d-fructose-6-phosphate aminotransferase), _glmU_ (encoding bifunctional N-acetylglucosamine-1-phosphate uridyltransferase and glucosamine-1-phosphate acetyltransferase), were

amplified from different species, including _E. coli_ MG1655 (ec), _P. putida_ KT2440 (pt), _S. equi_ subsp. _zooepidemicus_ (se) and _C. glutamicum_ ATCC13032 (cg), using the designated

primers listed in Supplementary Data 3. Additionally, we cloned the _ugdA_ genes located in the heparosan synthesis gene cluster of _E. coli_ O10:K5:H4 and _E. coli_ Nissle 1917. The primers

and the amplified genes were prefixed with “ec”, “pt”, “se”, “cg”, “eco”, and “ecn” in order to indicate their species origins. These genes were assembled individually or in specified

combinations into the pEC-XK99E plasmid using the TEDA method46. Homologous regions with a length of ~750 bp were amplified upstream and downstream of _cg0420_ and _cg0424_ using primers

0420-up-F/0420-up-R, 0420-down-F/0420-down-R, and 0424-up-F/0424-up-R, 0424-down-F/0424-down-R, respectively. The homologous regions were combined with the EcoRI/BamHI linearized pK18mobSacB

plasmid with to create pK18mobSacB-0420 and pK18mobSacB-0424, which were subsequently used for gene deletions of _cg0420_ and _cg0424_. All plasmids were transferred to _C. glutamicum_ ATCC

13032 by electroporation (200 Ω, 12.5 kV cm−1, pulse duration 4 ms) and a subsequent heat shock (46 °C for 6 minutes in BHIS medium)47. Recombinant strains were selected with kanamycin

resistance. In the same way, integrations of the plasmids pK18mobSacB-0420 and pK18mobSacB-0424 into the chromosome were selected with kanamycin resistance. Segregation of the plasmids from

chromosome was counter-selected with sucrose-induced lethality47. Loss of the gene _cg0420_ or _cg0424_ was confirmed with colony PCR and DNA sequencing. MEDIUM AND CULTIVATION _E. coli_ was

cultivated in Luria-Bertani medium (tryptone 10 g L−1, NaCl 10 g L−1, yeast extract 5 g L−1, pH 7.0) at 37 °C, and _C. glutamicum_ was cultivated at 30 °C in BHIS medium [37 g L−1 Brain

Heart Infusion (Difco), 91 g L−1 sorbitol]. Competent _C. glutamicum_ cells for electroporation were prepared by cultivating _C. glutamicum_ strains in BHIS medium to OD600 of 1.5 and

washing cells three times with 10% (v/v) glycerol47. Chloramphenicol (15 mg L−1), kanamycin (25 mg L−1) or sucrose [15% (m/v)] was supplemented, as necessary. To produce HA in shake-flasks,

engineered _C. glutamicum_ strains were cultivated at 28 °C with shaking at 200 rpm in a modified glucose-corn steep powder medium29. The medium contained 40 g L−1 glucose, 20 g L−1 corn

steep  powder, 20 g L−1 (NH4)2SO4, 1 g L−1 KH2PO4, 1 g L−1 K2HPO4, 0.25 g L−1 MgSO4, and 42 g L−1 MOPS [3-(N-morpholino) propanesulfonic acid]. The initial  optical density at 600 nm (OD600)

of the shake-flask cultivation was 0.2, and isopropyl‐β‐d‐thiogalactoside (IPTG) was added to induce target gene expression at time point 2.5 hour. LHYal was supplemented at time point 20 

hour to a final concentration of 6000 U mL−1, as necessary. Fed-batch cultivations of _C. glutamicum_ were performed in a 5-L fermenter with 2.5 L of the glucose-corn steep powder medium.

Seed cultures were prepared in glucose-corn steep powder medium containing 40 g L−1 glucose at 30 °C and shaken at 220 rpm for 8–10 hours. During fed-batch cultivations, glucose [70% (m/v)]

was fed according to real-time glucose consumption in order to dynamically maintain its concentration between 10 and 15 g L−1. Ammonia solution [14% (v/v)] was automatically fed in order to

maintain the culture pH at between 6.5 and 7.0. Air flow was maintained at ~5 vvm. LHYal was supplemented to final concentrations of 1500, 3000, or 6000 U mL−1 at time point 20 hour. _S.

equi_ subsp. _zooepidemicus _WSH-24 seed culture was grown at 37 °C and shaking at 220 rpm for 14–16 hours in medium containing 20 g L−1 glucose, 20 g L−1 yeast extract, 2 g L−1 MgSO4·7H2O,

0.1 g L−1 MnSO4·4H2O, 1.5 g L−1 Na2HPO4, 0.64 g L−1 NaH2PO4, 2.0 g L−1 KH2PO4, 0.5 g L−1 NaHCO3, 20 g L−1 CaCO3, 200 μg L−1 CaCl2, 46 μg L−1 ZnCl2, 19 μg L−1 CuSO4·5H2O, pH 7.2. Seed

cultures were inoculated into a 3-L fermenter containing 1.5 L of fermentation medium with a composition of 70 g L−1 glucose, 20 g L−1 yeast extract, 6.2 g L−1 Na2HPO4, 1.3 g L−1 K2SO4, 2 g 

L−1 MgSO4·7H2O, 200 μg L−1 CaCl2, 46 μg L−1 ZnCl2, 19 μg L−1 CuSO4·5H2O, pH 7.2. For fed-batch cultivation at 37 °C, NaOH (5 M) was automatically fed to maintain pH between 6.9 and 7.1, and

air flow was maintained at ~3 vvm. LHYal (final concentration 6000 U mL−1) was supplemented at the time of seed inoculation. MEASUREMENT OF CELL GROWTH AND METABOLITE CONCENTRATION Cell

growth was monitored by changes in the OD600. Real-time glucose concentration was measured using an M-100 biosensor analyzer (Shenzhen Siemantec Technology, Shenzhen, China). To measure

total HA, culture broths were autoclaved at 121 °C for 30 minutes. Cell debris was removed by centrifugation at 16,099 × _g_ for 15 minutes. Supernatants were pooled and mixed with four

volumes of ice-cold ethanol and HA was precipitated at −30 °C overnight. Insoluble fractions were subsequently collected by centrifugation at 16,099 × _g_ for 10 minutes. Residual ethanol

was evaporated at room temperature and the semi-dry insoluble fraction was dissolved in deionized water, whereas the water-insoluble fraction was removed by centrifugation. To remove as much

impurity, these steps were repeated three times more. After appropriate dilution (5–1000 fold, depending on HA concentration), HA content was determined via carbazole assay using glucuronic

acid as an external standard48. Supernatants from _C. glutamicum_ culture harboring the blank vector pXMJ19 or pXMJ19/pEC-XK99E were used as the negative control. To measure secreted HA,

cells were removed from the broth by centrifugation at 16,099 × _g_ for 15 minutes and HA was precipitated from the supernatant for measurement, as described above. ANALYSIS OF _C.

GLUTAMICUM_ EXTRACELLULAR POLYSACCHARIDES _C. glutamicum_ ATCC 13032 and strains lacking _cg0424_ or _cg0420_ and _cg0424_ were cultivated in CGXII minimal medium47 at 28 °C for 48 h. Cells

were shaken with glass beads at 200 rpm and room temperature for 1 h to release surface exposed materials including extracellular polysaccharides38. Cells were removed by centrifugation at

7155 × _g_ for 20 minutes. Supernatants were pooled and hydrolyzed with trifluoroacetic acid at 110 °C for 2 hours. Hydrolysates containing reducing sugars were dried in vacuum chamber at 65

 °C for 2 hours. Reducing sugars generated from hydrolysis of extracellular polysaccharides reacted with 1-phenyl-3-methyl-5-pyrazolone (PMP) at 70 °C for 60 minutes in the NaOH-methanol

solution to form the sugar-PMP49. The sugar constituent of _C. glutamicum_ extracellular polysaccharides was analyzed using Agilent 1200 HPLC system equipped with a SHISEIDO CAPCELL PAK C18

column (inner diameter 4.6 mm, length 250 mm, particle size 5 μm). Chemicals were separated in the column using a mobile phase of 0.1 M KH2PO4 (pH 6.8) [82% (v/v)] and acetonitrile [18%

(v/v)] with a flow rate of 1 mL min−1. Fractions were detected with absorbance at wavelength of 245 nm. PREPARATION OF RECOMBINANT LEECH HYALURONIDASE LHYal samples were prepared and

purified from recombinant _Pichia pastoris_ through Ni-sepharose affinity resin (HisTrap FF column, GE Healthcare)32. Purified LHYal was stored at −30 °C until use. Determination of the

LHYal glycoside hydrolase activity was performed routinely by the 3,5-dinitrosalicylic acid colorimetric quantification of enzymatically released reducing sugars32. WESTERN BLOT OF TYPE I

HYALURONAN SYNTHASE _C. glutamicum_ ATCC13032 expressing C terminally 6× histidine tagged spHasA, seHasA, suHasA, seHasAThr7-Met25 were digested with 20 mg mL−1 lysozyme at room temperature

for 2 hours and lysed with sonication in buffer containing 50 mM Tris-HCl, 5 mM ethylenediaminetetraacetic acid and 1 mM phenylmethanesulfonyl fluoride. Cell debris was removed by

centrifugation at 16,099 × _g_ for 15 minutes. After BCA protein concentration assay, equal amount (20 μg) of total protein samples were applied to SDS-PAGE. Afterwards, Western Blot was

performed with 1:5000 diluted  YTHXBio ZA004 His-tag mouse monoclonal antibody and 1:10000  diluted horseradish peroxidase labeled YTHXBio ZM03 goat anti-mouse IgG(H + L)-HRP) (YTHX

Biotechnology, Beijing, China). ANALYSIS OF CELL MORPHOLOGY BY MICROSCOPY For phase-contrast microscopy, cells were loaded on the thin layer of an agarose pad and visualized using an Eclipse

Ni-E microscope (Nikon, Tokyo, Japan) equipped with a module for phase-contrast microscopy. To visualize cell encapsulation, cells were negatively stained with 100 mg mL−1 water-soluble

nigrosin (Sangon Biotech, Shanghai, China), air dried and visualized with Eclipse Ni-E bright field microscope. Micrographs were processed with ImageJ50. DETERMINATION OF HA EFFECTS ON CELL

METABOLISM AND GROWTH _C. glutamicum_ ATCC 13032 was grown in glucose-corn steep powder medium for 10 hours, followed by centrifugation at 1789 × _g_ for 10 minutes and resuspension with

fresh glucose-corn steep powder medium containing 20 g L−1 glucose and designated concentrations (0, 3, 5, or 10 g L−1) of HA [obtained from Bloomage Biotechnology CO, LTD (Jinan, China)

with purity higher than 98%] or HA pretreated with 10,000 U mL−1 LHYal at room temperature for 8 hours. The initial OD600 was set to 10 and cell growth and glucose consumption were measured

every 2 hours. MEASUREMENT HA WEIGHT AVERAGE MW Supernatant containing HA was isolated, as described above, using ice-cold ethanol precipitation. The crude HA samples were subjected to

further purification with anion exchange chromatography via the ÄKTA avant 25 preparative chromatography system equipped with a HiPrep Q HP 16/10 (GE Healthcare, USA) column. Column was

equilibrated with 50 mM Tris-HCl (pH 8.0); fractions were gradient eluted with 0–200 mM NaCl and monitored with absorbance at wavelength of 210 nm. Fractions were collected, freeze-dried,

dissolved with appropriate amount of water and filtrated through a 0.22 μm membrane. The weight average MW of HA was measured using high-performance size-exclusion chromatography (HPSEC)

with multi-angle laser light scattering (MALLS) analysis. Briefly, 100 μL of HA sample was injected into an HPSEC-MALLS system (equipped with Waters 515 HPLC pump, Shodex OHpak SB-806HQ and

Shodex OHpak SB-804HQ column series, DAWN HELEOS II MALLS instrument and Optilab dRI detector), fractions were separated in the column series with the mobile phase of 0.02% (m/v) NaN3 at 25 

°C at a flow rate of 1.0 mL min−1. The average value of two measurements was used to calculate the final weight average MW of HA. REPORTING SUMMARY Further information on research design is

available in the Nature Research Reporting Summary linked to this article. DATA AVAILABILITY The authors declare that all data supporting the findings of this study are available within the

paper and its supplementary information files. The datasets generated and analyzed during the current study are also available from the corresponding author upon request. The source data

underlying Figures 1b–e, 2c–e, 3a, 4a, b, d, 5a, b, 6a–c, and Tables 1 and  2 as well as Supplementary Figures 2b, c, 4a–c, 6 and Supplementary Table 1 are provided as a Source Data file. 

Source data are provided with this paper. REFERENCES * Stecco, C. et al. Hyaluronan within fascia in the etiology of myofascial pain. _Surg. Radio. Anat._ 33, 891–896 (2011). Article  Google

Scholar  * Averbeck, M. et al. Differential regulation of hyaluronan metabolism in the epidermal and dermal compartments of human skin by UVB irradiation. _J. Invest. Dermatol._ 127,

687–697 (2007). Article  CAS  PubMed  Google Scholar  * Wessels, M. R., Moses, A. E., Goldberg, J. B. & DiCesare, T. J. Hyaluronic acid capsule is a virulence factor for mucoid group A

streptococci. _Proc. Natl Acad. Sci. USA_ 88, 8317–8321 (1991). Article  ADS  CAS  PubMed  PubMed Central  Google Scholar  * Pandit, K. K. & Smith, J. E. Capsular hyaluronic acid in

_Pasteurella multocida_ type A and its counterpart in type D. _Res. Vet. Sci._ 54, 20–24 (1993). Article  CAS  PubMed  Google Scholar  * Xu, X., Jha, A. K., Harrington, D. A., Farach-Carson,

M. C. & Jia, X. Hyaluronic acid-based hydrogels: from a natural polysaccharide to complex networks. _Soft Matter_ 8, 3280–3294 (2012). Article  ADS  CAS  PubMed  PubMed Central  Google

Scholar  * Walimbe, T., Panitch, A. & Sivasankar, P. M. A review of hyaluronic acid and hyaluronic acid-based hydrogels for vocal fold tissue engineering. _J. Voice_ 31, 416–423 (2017).

Article  PubMed  PubMed Central  Google Scholar  * Widjaja, L. K. et al. Hyaluronic acid-based nanocomposite hydrogels for ocular drug delivery applications. _J. Biomed. Mater. Res. A_ 102,

3056–3065 (2014). Article  PubMed  CAS  Google Scholar  * Kang, Z. et al. Bio-based strategies for producing glycosaminoglycans and their oligosaccharides. _Trends Biotechnol._ 36, 806–818

(2018). Article  CAS  PubMed  Google Scholar  * Liu, L., Liu, Y., Li, J., Du, G. & Chen, J. Microbial production of hyaluronic acid: current state, challenges, and perspectives. _Microb.

Cell Fact._ 10, 99 (2011). Article  CAS  PubMed  PubMed Central  Google Scholar  * Mao, Z., Shin, H. D. & Chen, R. A recombinant _E. coli_ bioprocess for hyaluronan synthesis. _Appl.

Microbiol. Biotechnol._ 84, 63–69 (2009). Article  CAS  PubMed  Google Scholar  * Yu, H. & Stephanopoulos, G. Metabolic engineering of _Escherichia coli_ for biosynthesis of hyaluronic

acid. _Metab. Eng._ 10, 24–32 (2008). Article  CAS  PubMed  Google Scholar  * Westbrook, A. W., Ren, X., Moo-Young, M. & Chou, C. P. Application of hydrocarbon and perfluorocarbon oxygen

vectors to enhance heterologous production of hyaluronic acid in engineered _Bacillus subtilis_. _Biotechnol. Bioeng._ 115, 1239–1252 (2018). Article  CAS  PubMed  Google Scholar  * Chien,

L. J. & Lee, C. K. Enhanced hyaluronic acid production in _Bacillus subtilis_ by coexpressing bacterial hemoglobin. _Biotechnol. Prog._ 23, 1017–1022 (2007). CAS  PubMed  Google Scholar

  * Jia, Y. et al. Metabolic engineering of _Bacillus subtilis_ for the efficient biosynthesis of uniform hyaluronic acid with controlled molecular weights. _Bioresour. Technol._ 132,

427–431 (2013). Article  CAS  PubMed  Google Scholar  * Jin, P., Kang, Z., Yuan, P., Du, G. & Chen, J. Production of specific-molecular-weight hyaluronan by metabolically engineered

_Bacillus subtilis_ 168. _Metab. Eng._ 35, 21–30 (2016). Article  CAS  PubMed  Google Scholar  * Widner, B. et al. Hyaluronic acid production in _Bacillus subtilis_. _Appl. Environ.

Microbiol._ 71, 3747–3752 (2005). Article  CAS  PubMed  PubMed Central  Google Scholar  * Sunguroglu, C., Sezgin, D. E., Aytar Celik, P. & Cabuk, A. Higher titer hyaluronic acid

production in recombinant _Lactococcus lactis_. _Prep. Biochem. Biotechnol._ 48, 734–742 (2018). Article  CAS  PubMed  Google Scholar  * Sheng, J., Ling, P. & Wang, F. Constructing a

recombinant hyaluronic acid biosynthesis operon and producing food-grade hyaluronic acid in _Lactococcus lactis_. _J. Ind. Microbiol. Biotechnol._ 42, 197–206 (2015). Article  CAS  PubMed 

Google Scholar  * Jeeva, P., Shanmuga Doss, S., Sundaram, V. & Jayaraman, G. Production of controlled molecular weight hyaluronic acid by glucostat strategy using recombinant

_Lactococcus lactis_ cultures. _Appl. Microbiol. Biotechnol._ 103, 4363–4375 (2019). Article  CAS  PubMed  Google Scholar  * Hmar, R. V., Prasad, S. B., Jayaraman, G. & Ramachandran, K.

B. Chromosomal integration of hyaluronic acid synthesis (has) genes enhances the molecular weight of hyaluronan produced in _Lactococcus lactis_. _Biotechnol. J._ 9, 1554–1564 (2014).

Article  CAS  PubMed  Google Scholar  * Hoffmann, J. & Altenbuchner, J. Hyaluronic acid production with _Corynebacterium glutamicum_: effect of media composition on yield and molecular

weight. _J. Appl. Microbiol._ 117, 663–678 (2014). Article  CAS  PubMed  Google Scholar  * Cheng, F., Yu, H. & Stephanopoulos, G. Engineering _Corynebacterium glutamicum_ for high-titer

biosynthesis of hyaluronic acid. _Metab. Eng._ 55, 276–289 (2019). Article  CAS  PubMed  Google Scholar  * Jeong, E., Shim, W. Y. & Kim, J. H. Metabolic engineering of _Pichia pastoris_

for production of hyaluronic acid with high molecular weight. _J. Biotechnol._ 185, 28–36 (2014). Article  CAS  PubMed  Google Scholar  * Mandawe, J. et al. Directed evolution of hyaluronic

acid synthase from _Pasteurella multocida_ towards high-molecular-weight hyaluronic acid. _Chembiochem_ 19, 1414–1423 (2018). Article  CAS  PubMed  Google Scholar  * Jing, W. &

DeAngelis, P. L. Synchronized chemoenzymatic synthesis of monodisperse hyaluronan polymers. _J. Biol. Chem._ 279, 42345–42349 (2004). Article  CAS  PubMed  Google Scholar  * Schulte, S. et

al. Exploiting the diversity of streptococcal hyaluronan synthases for the production of molecular weight-tailored hyaluronan. _Appl. Microbiol. Biotechnol._ 103, 7567–7581 (2019). Article 

CAS  PubMed  Google Scholar  * Baggenstoss, B. A. et al. Hyaluronan synthase control of synthesis rate and hyaluronan product size are independent functions differentially affected by

mutations in a conserved tandem B-X7-B motif. _Glycobiology_ 27, 154–164 (2017). Article  CAS  PubMed  PubMed Central  Google Scholar  * Westbrook, A. W., Ren, X., Moo-Young, M. & Chou,

C. P. Engineering of cell membrane to enhance heterologous production of hyaluronic acid in _Bacillus subtilis_. _Biotechnol. Bioeng._ 115, 216–231 (2018). Article  CAS  PubMed  Google

Scholar  * Cheng, F., Luozhong, S., Guo, Z., Yu, H. & Stephanopoulos, G. Enhanced biosynthesis of hyaluronic acid using engineered _Corynebacterium glutamicum_ via metabolic pathway

regulation. _Biotechnol. J._ 12, 1700191 (2017). Article  CAS  Google Scholar  * Kaur, M. & Jayaraman, G. Hyaluronan production and molecular weight is enhanced in pathway-engineered

strains of lactate dehydrogenase-deficient _Lactococcus lactis_. _Metab. Eng. Commun._ 3, 15–23 (2016). Article  PubMed  PubMed Central  Google Scholar  * Cheng, F., Gong, Q., Yu, H. &

Stephanopoulos, G. High-titer biosynthesis of hyaluronic acid by recombinant _Corynebacterium glutamicum_. _Biotechnol. J._ 11, 574–584 (2016). Article  CAS  PubMed  Google Scholar  * Jin,

P., Kang, Z., Zhang, N., Du, G. & Chen, J. High-yield novel leech hyaluronidase to expedite the preparation of specific hyaluronan oligomers. _Sci. Rep._

https://doi.org/10.1038/srep04471 (2014). * Weigel, P. H. Hyaluronan synthase: the mechanism of initiation at the reducing end and a pendulum model for polysaccharide translocation to the

cell exterior. _Int. J. Cell Biol._ 2015, 367579 (2015). * DeAngelis, P. L., Papaconstantinou, J. & Weigel, P. H. Isolation of a _Streptococcus pyogenes_ gene locus that directs

hyaluronan biosynthesis in acapsular mutants and in heterologous bacteria. _J. Biol. Chem._ 268, 14568–14571 (1993). CAS  PubMed  Google Scholar  * Heldermon, C., DeAngelis, P. L. &

Weigel, P. H. Topological organization of the hyaluronan synthase from _Streptococcus pyogenes_. _J. Biol. Chem._ 276, 2037–2046 (2001). Article  CAS  PubMed  Google Scholar  * DeAngelis, P.

L., Jing, W., Drake, R. R. & Achyuthan, A. M. Identification and molecular cloning of a unique hyaluronan synthase from _Pasteurella multocida_. _J. Biol. Chem._ 273, 8454–8458 (1998).

Article  CAS  PubMed  Google Scholar  * Donot, F., Fontana, A., Baccou, J. C. & Schorr-Galindo, S. Microbial exopolysaccharides: main examples of synthesis, excretion, genetics and

extraction. _Carbohydr. Polym._ 87, 951–962 (2012). Article  CAS  Google Scholar  * Puech, V. et al. Structure of the cell envelope of corynebacteria: importance of the non-covalently bound

lipids in the formation of the cell wall permeability barrier and fracture plane. _Microbiology_ 147, 1365–1382 (2001). Article  CAS  PubMed  Google Scholar  * Jackson, M. The mycobacterial

cell envelope-lipids. _Cold Spring Harb. Perspect. Med._ https://doi.org/10.1101/cshperspect.a021105 (2014). * Taniguchi, H. et al. Physiological roles of sigma factor SigD in

_Corynebacterium glutamicum_. _BMC Microbiol._ 17, 158 (2017). Article  PubMed  PubMed Central  CAS  Google Scholar  * Islam, S. T. & Lam, J. S. Synthesis of bacterial polysaccharides

via the Wzx/Wzy-dependent pathway. _Can. J. Microbiol._ 60, 697–716 (2014). Article  CAS  PubMed  Google Scholar  * Taubitz, I. S. & Brandis, H. A comparison between methods of

identification and serotyping of encapsulated strains of _Haemophilus influenzae_. _Zentralblatt fur Bakteriologie, Mikrobiologie, und Hyg._ 270, 83–97 (1988). CAS  Google Scholar  * Yu, H.,

Tyo, K., Alper, H., Klein-Marcuschamer, D. & Stephanopoulos, G. A high-throughput screen for hyaluronic acid accumulation in recombinant _Escherichia coli_ transformed by libraries of

engineered sigma factors. _Biotechnol. Bioeng._ 101, 788–796 (2008). Article  CAS  PubMed  Google Scholar  * Zhang, L. et al. Rapid evolution of hyaluronan synthase to improve hyaluronan

production and molecular mass in _Bacillus subtilis_. _Biotechnol. Lett._ 38, 2103–2108 (2016). Article  CAS  PubMed  Google Scholar  * Tzeng, Y. L., Thomas, J. & Stephens, D. S.

Regulation of capsule in _Neisseria meningitidis_. _Crit. Rev. Microbiol_ 42, 759–772 (2016). CAS  PubMed  Google Scholar  * Xia, Y. et al. T5 exonuclease-dependent assembly offers a

low-cost method for efficient cloning and site-directed mutagenesis. _Nucleic Acids Res._ https://doi.org/10.1093/nar/gky1169 (2019). * Eggeling, L. & Bott, M. _Handbook of

Corynebacterium glutamicum_. (Taylor & Francis, 2005). * Bitter, T. & Muir, H. M. A modified uronic acid carbazole reaction. _Anal. Biochem._ 4, 330–334 (1962). Article  CAS  PubMed

  Google Scholar  * Wang, W. et al. Optimization of reactions between reducing sugars and 1-phenyl-3-methyl-5-pyrazolone (PMP) by response surface methodology. _Food Chem._ 254, 158–164

(2018). Article  CAS  PubMed  Google Scholar  * Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. _Nat. Methods_ 9, 676–682 (2012). Article  CAS  PubMed 

Google Scholar  Download references ACKNOWLEDGEMENTS This work was financially supported by the National Key R&D program of China (2018YFA0901401), the National Natural Science

Foundation of China (31970085), a grant from the Key Technologies R&D Program of Jiangsu Province (BE2019630), the Fundamental Research Funds for the Central Universities (JUSRP51707A),

the Program for Changjiang Scholars and Innovative Research Team in University (IRT_15R26), and the 111 Project. Y.W. was sponsored by the China Postdoctoral Science Foundation

(2019M651702). AUTHOR INFORMATION Author notes * These authors contributed equally: Yang Wang, Litao Hu. AUTHORS AND AFFILIATIONS * The Key Laboratory of Carbohydrate Chemistry and

Biotechnology, Ministry of Education, Jiangnan University, 214122, Wuxi, China Yang Wang, Litao Hu, Jian Chen, Guocheng Du & Zhen Kang * The Key Laboratory of Industrial Biotechnology,

Ministry of Education, School of Biotechnology, Jiangnan University, 214122, Wuxi, China Yang Wang, Litao Hu, Hao Huang, Hao Wang, Jian Chen, Guocheng Du & Zhen Kang * Bloomage

Biotechnology CO, LTD, 250000, Jinan, China Tianmeng Zhang Authors * Yang Wang View author publications You can also search for this author inPubMed Google Scholar * Litao Hu View author

publications You can also search for this author inPubMed Google Scholar * Hao Huang View author publications You can also search for this author inPubMed Google Scholar * Hao Wang View

author publications You can also search for this author inPubMed Google Scholar * Tianmeng Zhang View author publications You can also search for this author inPubMed Google Scholar * Jian

Chen View author publications You can also search for this author inPubMed Google Scholar * Guocheng Du View author publications You can also search for this author inPubMed Google Scholar *

Zhen Kang View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS Y.W. and L.H. performed the experiments. Z.K. and Y.W. designed the experiments.

H.H. purified the leech hyaluronidase. H.W. contributed to the shake-flask cultivations. T.Z. contributed to the fed-batch cultivation. Z.K., Y.W., J.C. and G.D. drafted the manuscript.

CORRESPONDING AUTHOR Correspondence to Zhen Kang. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. ADDITIONAL INFORMATION PEER REVIEW INFORMATION _Nature

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layer to promote glucose uptake for hyaluronan production by engineered _Corynebacterium glutamicum_. _Nat Commun_ 11, 3120 (2020). https://doi.org/10.1038/s41467-020-16962-7 Download

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