Bacterial protein secretion represents a significant challenge in biotechnology, which is essential for the cost‐effective production of therapeutics, enzymes, and other functional proteins. Here, it is demonstrated that proteomics‐guided engineering of transcription, translation, secretion, and folding of ligninolytic laccase balances the process, minimizes the toxicity, and enables efficient heterologous secretion with a total protein yield of 13.7 g L
−1
. The secretory laccase complements the biochemical limits on lignin depolymerization well in
Rhodococcus opacus
PD630. Further proteomics analysis reveals the mechanisms for the oleaginous phenotype of
R. opacus
PD630, where a distinct multiunit fatty acid synthase I drives the carbon partition to storage lipid. The discovery guides the design of efficient lipid conversion from lignin and carbohydrate. The proteomics‐guided integration of laccase‐secretion and lipid production modules enables a high titer in converting lignin‐enriched biorefinery waste to lipid. The fundamental mechanisms, engineering components, and design principle can empower transformative platforms for biomanufacturing and biorefining.
Inadequate disposal of Agave tequilana bagasse
(ATB) brings environmental and economic issues to tequila producing
regions. Recent works have proposed technologies for valorization
of the polysaccharide’s fractions of ATB. However, lignin bioconversion
has not been investigated due to its recalcitrant nature. Herein,
a systematic pretreatment is proposed to release the lignocellulosic
fractions and produce medium-chain-length polyhydroxyalkanoates (PHA)
from the lignin fraction of ATB. Nuclear magnetic resonance (NMR)
analyses revealed that ATB lignin contained 63% of β-O-4 interunit
linkages, making this lignin more suitable for pretreatment and biodegradation
compared to other lignins. Exploratory experiments revealed that two-stage
fermentation is suitable for PHA production from ATB lignin using
wild type and engineered Pseudomonas putida strains.
PHA increased from 0.09 to 0.39 g/L compared to single batch fermentation.
Further improvement to 0.76 g/L was possible by using a central composite
design to optimize the inoculum, substrate, and nitrogen concentrations.
Finally, PHA depolymerase gene phaZ was knocked out
from P. putida and tested at optimal conditions,
enabling a PHA titer of 0.97 g/L. Analyses by NMR and GC-MS confirmed
lignin derivatives consumption and decrease of β-O-4 linkages.
This study lays the foundations to enable agave lignin bioprocessing
and opens new avenues for ATB biorefineries.
Pelletized
liquid cultivation has been widely explored because
of its advantages in biomanufacturing, such as easier biomass harvesting,
higher product yield, and lower medium viscosity and energy consumption.
In this study, we discovered that the nonfilamentous bacterium Rhodococcus opacus PD630 could form pellets during
the fermentation of alkaline pretreatment liquor containing lignin
as a carbon source. This discovery advanced our understanding of bacterium
pelletization, as only filamentous fungi and filamentous bacteria
were reported to form pellets without the addition of external agents
such as flocculants or polymers in previous research. Several factors
were investigated to understand how they affect the process of pelletization.
Notably, the lipid content in the pellets was much higher than in
the scattered bacteria at low nitrogen concentration (<0.5 g/L),
under which condition (high carbon to nitrogen ratio) the industrial
microbial production for lipids was carried out. Moreover, the highest
pellet percentage (∼60% of the total biomass) was observed
at 30 g/L soluble solid content, an agitation rate of 180 rpm, 1.4
g/L NH4NO3, an initial optical density (OD600) of 10, and a centrifugation speed of 6000 rpm. The study
also opens new avenues to decrease harvesting and cultivation cost
as well as energy consumption for microbial fermentation.
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