High-Efficiency Secretion of β-Mannanase in Bacillus subtilis through Protein Synthesis and Secretion Optimization

Abstract

The manno endo-1,4-mannosidase (β-mannanase, EC. 3.2.1.78) catalyzes the random hydrolysis of internal (1 → 4)-β-mannosidic linkages in the mannan polymers. A codon optimized β-mannanase gene from Bacillus licheniformis DSM13 was expressed in Bacillus subtilis. When four Sec-dependent and two Tat-dependent signal peptide sequences cloned from B. subtilis were placed upstream of the target gene, the highest activity of β-mannanase was observed using SP as a signal peptide. Then a 1.25-fold activity of β-mannanase was obtained when another copy of groESL operon was inserted into the genome of host strain. Finally, five different promoters were separately used to enhance the synthesis of the target protein. The results showed that promoter P, a modified maltose-inducible promoter, significantly elevated the production of β-mannanase. After 72 h of flask fermentation, the enzyme activity of β-mannanase in the supernatant when using locust bean gum as substrate reached 2207 U/mL. This work provided a promising β-mannanase production strain in industrial application.

EngineeringBacillusfor efficient production of heterologous protein: current progress, challenge and prospect

“…Generally, two types of intracellular molecular chaperones GroES and DnaK exist in Bacillus , and PrsA is a well‐defined extracellular lipoprotein required for subsequent folding of mature proteins (Song et al . ). The formation of an inclusion body was decreased and secretory signal chain antibody yield was enhanced in the chaperones GroES, DnaK and PrsA overexpression strain (Wu et al .…”

“…Previously, overexpression of type I signal peptidase SipM significantly enhanced the yield of recombinant dextransucrase DsrS from Leuconostoc mesenteroides in Bacillus megaterium MS941 (Malten et al 2005). Similarly, overexpression of signal peptidases SipS and SipT promoted b-mannanase activity (Song et al 2017), and strengthening SipT benefited preAmyQ production in B. subtilis (Pummi et al 2002). In addition, the nattokinase activity was reduced by 45% in the signal peptidase gene sipV deletion strain, and overexpression of SipV led to a 16% increase of nattokinase production in B. licheniformis (Cai et al 2016).…”

“…Similarly, overexpression of signal peptidases SipS and SipT promoted β‐mannanase activity (Song et al . ), and strengthening SipT benefited preAmyQ production in B. subtilis (Pummi et al . ).…”

“…Recently, several tactics have been conducted to improve the target protein production by strengthening Sec pathway. For instance, overexpression of transportation elements (SecA, SecDF, SecYEG, SRP and FtsY) effectively improves the activity of a-amylase (Chen et al 2015c), as well as those of b-mannanase (Song et al 2017) and lipase . Acting as a scissor, signal peptidase cleaves away the signal peptide to form a mature protein ( van Dijl 2012), and the overexpression of signal peptidase can enhance the shear efficiency of the signal peptide from a preprotein, which further enhances the secretion efficiency of the target protein (Malten et al 2005).…”

“…The low yield of heterologous proteins from Bacillus protein expression systems hindered its application, and (Zhou et al 2017;Huang et al 2018), signal peptides (Cai et al 2016), transportation elements (Chen et al 2015c), signal peptidases (Cai et al 2016;Song et al 2017), signal peptide peptidases , chaperones (Chen et al 2015d), proteases (Pummi et al 2002;Wei et al 2015), transcriptional factors ), etc, have been genetically modified for enhanced production of target proteins (a-amylase, protease, pullulanase, etc. ), also, the nonclassical secretion pathways have been developed and applied for protein production in B. subtilis Zhao et al 2017).…”