Abstract
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.
Highlights
Bacterial protein secretion represents a significant challenge in biotechnology, available genetic toolkit.[1]
The study revealed several aspects of fundamental mechanisms regarding protein secretion, lipid biosynthesis, and aromatic utilization in bacteria, which further guided the biodesign of efficient secretory protein production and lignin conversion
On one side, the results suggested that the key for efficient secretion of heterologous protein lies in the balance of secretion and transcription
Summary
Considering the strong capacity for aromatic compound degradation, we will focus on designing the functional modules for laccase secretion and lipid production to achieve more efficient lignin bioconversion. Proteomics analysis under different carbon sources revealed a distinct expression pattern for lipid biosynthesis enzymes, where Type I system (FASI) was significantly upregulated at the stationary phase in glucose medium, when lipid accumulation was stimulated (Figure 4a,b, and Figure S11, Supporting Information). Similar with the cell growth, the laccase activity of strain PD630_FL was increasing until the fourth day and significantly decreased, which might be caused by either laccase absorption to lignin or the loss of laccase expression module in bacteria (Figure S15, Supporting Information). Even higher lipid production at 2.54 ± 0.22 g L−1 could be achieved at day six using fed-batch fermentation with biorefinery waste from AFEX pretreated corn stover, representing the record bioproduct titer from biorefinery waste (Figure 6c)
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