Production of specific-molecular-weight hyaluronan by metabolically engineered Bacillus subtilis 168

Jin, Peng; Kang, Zhen; Yuan, Panhong; Du, Guocheng; Chen, Jian

doi:10.1016/j.ymben.2016.01.008

Cited by 121 publications

(76 citation statements)

References 69 publications

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“…A significantly longer cultivation was required to achieve a similar titer, and molecular mass was not assessed in the aforementioned study. In addition, the titer and molecular mass of HA produced by AW005-2 were similar to those obtained with strains of B. subtilis overexpressing additional enzymes of the HA biosynthetic pathway (i.e., GtaB, GlmM, GlmS, and GlmU) in combination with HasA and TuaD (69). Accordingly, it appears that chromosomal expression of Cas9 does not hinder HA production, and this feature is critical for the tool kit to be applied to industrial strain development.…”

Section: Figsupporting

confidence: 58%

Development of a CRISPR-Cas9 Tool Kit for Comprehensive Engineering of Bacillus subtilis

Westbrook

Moo‐Young

Chou

2016

Appl Environ Microbiol

165

156

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The establishment of a clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system for strain construction in Bacillus subtilis is essential for its progression toward industrial utility. Here we outline the development of a CRISPR-Cas9 tool kit for comprehensive genetic engineering in B. subtilis. In addition to site-specific mutation and gene insertion, our approach enables continuous genome editing and multiplexing and is extended to CRISPR interference (CRISPRi) for transcriptional modulation. Our tool kit employs chromosomal expression of Cas9 and chromosomal transcription of guide RNAs (gRNAs) using a gRNA transcription cassette and counterselectable gRNA delivery vectors. Our design obviates the need for multicopy plasmids, which can be unstable and impede cell viability. Efficiencies of up to 100% and 85% were obtained for single and double gene mutations, respectively. Also, a 2.9-kb hyaluronic acid (HA) biosynthetic operon was chromosomally inserted with an efficiency of 69%. Furthermore, repression of a heterologous reporter gene was achieved, demonstrating the versatility of the tool kit. The performance of our tool kit is comparable with those of systems developed for Escherichia coli and Saccharomyces cerevisiae, which rely on replicating vectors to implement CRISPR-Cas9 machinery. IMPORTANCEIn this paper, as the first approach, we report implementation of the CRISPR-Cas9 system in Bacillus subtilis, which is recognized as a valuable host system for biomanufacturing. The study enables comprehensive engineering of B. subtilis strains with virtually any desired genotypes/phenotypes and biochemical properties for extensive industrial application.

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Section: Figsupporting

confidence: 58%

Development of a CRISPR-Cas9 Tool Kit for Comprehensive Engineering of Bacillus subtilis

Westbrook

Moo‐Young

Chou

2016

Appl Environ Microbiol

165

156

View full text Add to dashboard Cite

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“…A recent study revealed the effects of overexpressing various combinations of genes involved in the HA biosynthetic pathway, including szHas, tuaD, gtaB, gcaD, glmS (encoding native L-glutamine-Dfructose-6-phosphate amidotransferase; GlmS), and glmM (encoding native phosphoglucosamine mutase; GlmM), as well as downregulating expression of pfkA (encoding 6-phosphofructokinase; PfkA) on HA MW and titer (Jin, Kang, Yuan, Du, & Chen, 2016). On the other hand, simply downregulating expression of pfkA, via replacement of the native ATG start codon with TTG, improved the HA titer by 18% (Jin et al, 2016). On the other hand, simply downregulating expression of pfkA, via replacement of the native ATG start codon with TTG, improved the HA titer by 18% (Jin et al, 2016).…”

Section: Introductionmentioning

confidence: 99%

“…Significant HA production can be achieved in strains of B. subtilis coexpressing only HAS and TuaD (Chien & Lee, 2007a;Jin et al, 2016;Widner et al, 2005), for which the expression is repressed in the presence of excess phosphate (Soldo, Lazarevic, Pagni, & Karamata, 1999). Accordingly, an HA-producing control strain was constructed by transforming B. subtilis 168 with NdeI-linearized pAW008 (Westbrook et al, 2016), a vector previously constructed for the integration of a P grac ::seHas:tuaD cassette at the amyE locus (Figure 1), yielding strain AW006 with a prominent mucoid phenotype characteristic of HA production in B. subtilis (Widner et al, 2005).…”

mentioning

confidence: 99%

Engineering of cell membrane to enhance heterologous production of hyaluronic acid in Bacillus subtilis

Westbrook

Ren

Moo‐Young

et al. 2017

Biotech & Bioengineering

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Hyaluronic acid (HA) is a high-value biopolymer used in the biomedical, pharmaceutical, cosmetic, and food industries. Current methods of HA production, including extraction from animal sources and streptococcal cultivations, are associated with high costs and health risks. Accordingly, the development of bioprocesses for HA production centered on robust "Generally Recognized as Safe (GRAS)" organisms such as Bacillus subtilis is highly attractive. Here, we report the development of novel strains of B. subtilis in which the membrane cardiolipin (CL) content and distribution has been engineered to enhance the functional expression of heterologously expressed hyaluronan synthase (HAS) of Streptococcus equisimilis (SeHAS), in turn, improving the culture performance for HA production. Elevation of membrane CL levels via overexpressing components involved in the CL biosynthesis pathway, and redistribution of CL along the lateral membrane via repression of the cell division initiator protein FtsZ resulted in increases to the HA titer of up to 204% and peak molecular weight of up to 2.2 MDa. Moreover, removal of phosphatidylethanolamine and neutral glycolipids from the membrane of HA-producing B. subtilis via inactivation of pssA and ugtP, respectively, has suggested the lipid dependence for functional expression of SeHAS. Our study demonstrates successful application of membrane engineering strategies to develop an effective platform for biomanufacturing of HA with B. subtilis strains expressing Class I streptococcal HAS.

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“…This suggests that both strains utilize sucrose more efficiently than wild type for HA biosynthesis. Recently, it is reported that down-regulation the expression of pfkA , a gene encoding phosphofructokinase, increase the HA yield in Bacillus subtilis (Jin et al 2016). Here, we found that deletion of pfk in S. zooepidemicus results in inhibition of the growth and HA production (Fig.…”

Section: Discussionmentioning

confidence: 99%

Construction of efficient Streptococcus zooepidemicus strains for hyaluoronic acid production based on identification of key genes involved in sucrose metabolism

Zhang

Wang

et al. 2016

AMB Expr

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Biosynthesis of polysaccharide hyaluoronic acid (HA) by Streptococcus zooepidemicus is a carbon-intensive process. The carbon flux and factor(s) restricting HA yield were not well understood. Here, we investigated the function of genes involved in sucrose metabolism and identified targets limiting HA yield, which were exploited to construct efficient S. zooepidemicus strains for HA production. The sucrose uptake was addressed by deletion of scrA and scrB, which encodes sucrose-PTS permease and sucrose-6-phosphate hydrolase, respectively. We found that scrB was essential for the growth of S. zooepidemicus and HA biosynthesis, and accumulation of sucrose-6-phosphate was toxic. ΔscrB could not grow in THY-sucrose medium, while ΔscrA and ΔscrAΔscrB showed negligible growth defects. Overexpression of scrA significantly reduced biomass and HA production, while overexpression of scrB resulted in 26% increase of biomass and 30% increase of HA yield. We revealed that fructose-6-phosphate for HA biosynthesis mainly originates from glucose-6-phosphate. Deletion of scrK, a gene encoding hexokinase, led to 11% reduction of biomass and 12% decrease of HA yield, while deletion of hasE, a gene encoding phosphoglucoisomerase, resulted in the abolishment of HA biosynthesis and a significantly slow growth. We found that HA biosynthesis could be improved by directing carbon flux to fructose-6-phosphate. Deletion of fruA encoding the EII of fructose-PTS and fruK encoding phosphofructokinase showed no apparent effect on cell growth, but resulted in 22 and 27% increase of HA yield, respectively. Finally, a strain with 55% increase of HA was constructed by overexpression of scrB in ΔfruK. These results provide a solid foundation for further metabolic engineering of S. zooepidemicus for highly efficient HA production.Electronic supplementary materialThe online version of this article (doi:10.1186/s13568-016-0296-7) contains supplementary material, which is available to authorized users.

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Production of specific-molecular-weight hyaluronan by metabolically engineered Bacillus subtilis 168

Cited by 121 publications

References 69 publications

Development of a CRISPR-Cas9 Tool Kit for Comprehensive Engineering of Bacillus subtilis

Development of a CRISPR-Cas9 Tool Kit for Comprehensive Engineering of Bacillus subtilis

Engineering of cell membrane to enhance heterologous production of hyaluronic acid in Bacillus subtilis

Construction of efficient Streptococcus zooepidemicus strains for hyaluoronic acid production based on identification of key genes involved in sucrose metabolism

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