Search bioRxiv⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/2024.07.07.602432

Improving Mannanase Production in Bacillus subtilis for Fibre Hydrolysis during Solid-State Fermentation of Palm Kernel Meal

Ong, W. L.·Li, Z.·Ng, K. H.·Zhou, K.
View original source ↗Export metadata ↓

Abstract

The primary challenge in utilizing palm kernel meal (PKM, an agricultural by-product) as non- ruminant livestock feed is its high fibre content, predominantly in the form of mannan. Microbial fermentation offers an economically favourable alternative to enzyme supplementation for breaking down fibre in lignocellulosic biomass. In a recent study, we have isolated and characterized an undomesticated strain (Bacillus subtilis F6) that is able to secrete mannanase. In this work, the mannanase production was substantially improved by optimizing multiple regulatory elements controlling the mannanase expression. Mannanase GmuG, sourced from B. subtilis F6 and verified for its hydrolytic activity on PKM fibre, was expressed using a replicative plasmid (pBE-S). The recombinant strain of B. subtilis F6 exhibited 1.9-fold increase in the mannanase activity during solid-state fermentation. Optimization of signal peptide and ribosome binding site further enhanced mannanase activity by 3.1-fold. Subsequently, promoter screening based on highly transcribed genes in B. subtilis F6 resulted in a significant 5.4-fold improvement in mannanase activity under the nprE promoter. The nprE promoter was further refined by eliminating specific transcription factor binding sites, enhancing the mannanase activity further by 1.8-fold. Notably, a substantial 35-40% reduction in PKM fibre content was observed after 30 h of fermentation using the recombinant strains. Lastly, the highest mannanase-producing strain was examined for scaled-up fermentation. The impacts of fermentation on fibre and protein contents, as well as the surface morphology of PKM, were analysed. The outcomes of this study offer an efficient method for robust mannanase expression in B. subtilis and its potential application in the biotransformation of PKM and other mannan-rich bioresources for improved feed utilization. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=83 SRC="FIGDIR/small/602432v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@10fbb9corg.highwire.dtl.DTLVardef@1e619fborg.highwire.dtl.DTLVardef@1b3bc0corg.highwire.dtl.DTLVardef@fec816_HPS_FORMAT_FIGEXP M_FIG C_FIG

Explore related subjects

synthetic biologyPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Ong, W. L., Li, Z., Ng, K. H., Zhou, K.. 2024-07-08. Improving Mannanase Production in Bacillus subtilis for Fibre Hydrolysis during Solid-State Fermentation of Palm Kernel Meal. https://doi.org/10.1101/2024.07.07.602432

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Coupled enzyme discovery, evolution and synthetic yeast chassis adaptation for microbial biopolymer valorisation

The valorisation of biological polymers requires microbial systems that can both access recalcitrant substrates and convert the resulting carbon into useful products. Although microbial genome and metagenome resources provide an expanding reservoir of candidate depolymerizing and modifying enzymes, most discovery workflows remain disconnected from enzyme optimisation and host adaptation. Here we present a coupled sequence-based enzyme discovery, enzyme evolution and synthetic yeast chassis adaptation strategy for microbial biopolymer valorisation. Focusing on laccases for the depolymerisation of lignin as a proof of concept, we combine sequence data mining for enzyme discovery, modular yeast surface display for functional screening, directed evolution for enzyme optimisation and synthetic yeast genome diversification for chassis improvement. In our study, surface display enabled functional benchmarking and recovery of improved laccase variants and synthetic-genome-enabled diversification provided a route to explore host configurations that influence display and enzyme performance. By integrating enzyme-level and chassis-level optimisation, this framework addresses a central bottleneck in converting microbial biodiversity by computational sequence repository mining into deployable biomanufacturing systems. Our results establish laccases as tractable entry points for oxidative biopolymer conversion and provide a generalizable platform for engineering yeast systems for sustainable carbon valorisation.

synthetic biology↗
bioRxiv2026

Multichromatic Dynamic Control of Multi-Membered Microbial Consortia Compositions for Chemical Production

Engineered microbial consortia offer a promising strategy for chemical production by distributing specialized functions among microbial strains, reducing metabolic burden, facilitating modular pathway optimization, reducing toxicity, and increasing strain stability. However, differences in growth rates can destabilize population composition, compromising productivity and limiting their applicability. Here, we developed a multichromatic optogenetic Toxin-Antitoxin (optogeneticTA) platform for dynamic control of Escherichia coli consortia of up to four members using blue, red, and near-infrared light and darkness. By varying light intensities or pulses, we precisely program and dynamically modulate the population composition of two-, three-, and four-membered consortia. We further developed a modular mathematical framework that captures and predicts population dynamics of these optogenetically controlled co-cultures. Applying dynamic control to a two-membered engineered consortium increased phenol production by ~69% relative to unregulated consortia. These results establish a programmable platform for stabilizing and dynamically optimizing microbial consortia, with potential applications across microbial biomanufacturing.

synthetic biology↗
bioRxiv2026

Reassessing the contribution of the histone H3 tail to KRAB-DNMT3L-mediated epigenetic silencing

Neumann et al. introduced CHARM, a compact epigenetic silencer in which a histone H3 tail fused to DNMT3L was proposed to recruit and stimulate endogenous DNMT3A, enabling durable gene repression without a fused DNMT3A catalytic domain. Here, we evaluated the contribution of the H3 tail in independent reporter and endogenous-gene contexts. In an SNRPN reporter system, a KRAB-DNMT3L-dCas9 construct lacking the H3 tail displayed silencing kinetics comparable to CRISPRcharm Kv2, and mutating the critical H3K4 residue to alanine in CRISPRcharm Kv2 did not compromise this silencing. Similarly, after transient delivery of editor mRNAs to HEK293T cells, CRISPRcharm Kv2 did not consistently outperform the corresponding H3-tail-free construct at three endogenous loci, and mutating the critical H3K4 residue to alanine in CRISPRcharm Kv2 did not compromise this activity. These observations suggest that the engineered H3 tail does not confer a general functional advantage within the KRAB-DNMT3L-dCas9 architecture under the conditions tested.

synthetic biology↗