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Microbial production of intracellular compounds can be engineered by redirecting the carbon flux towards products and increasing the cell size. Potential engineering strategies include exploiting clustered regularly interspaced short palindromic repeats interference (CRISPRi)‐based tools for controlling gene expression. Here, we applied CRISPRi for engineering Acinetobacter baylyi ADP1, a model bacterium for synthesizing intracellular storage lipids, namely wax esters. We first established an inducible CRISPRi system for strain ADP1, which enables tightly controlled repression of target genes. We then targeted the glyoxylate shunt to redirect carbon flow towards wax esters. Second, we successfully employed CRISPRi for modifying cell morphology by repressing ftsZ, an essential gene required for cell division, in combination with targeted knock‐outs to generate significantly enlarged filamentous or spherical cells respectively. The engineered cells sustained increased wax ester production metrics, demonstrating the potential of cell morphology engineering in the production of intracellular lipids. [ABSTRACT FROM AUTHOR]

Microbial storage compounds, such as wax esters (WE), are potential high‐value lipids for the production of specialty chemicals and medicines. Their synthesis, however, is strictly regulated and competes with cell growth, which leads to trade‐offs between biomass and product formation. Here, we use metabolic engineering and synergistic substrate cofeeding to partition the metabolism of Acinetobacter baylyi ADP1 into two distinct modules, each dedicated to cell growth and WE biosynthesis, respectively. We first blocked the glyoxylate shunt and upregulated the WE synthesis pathway to direct the acetate substrate exclusively for WE synthesis, then we controlled the supply of gluconate so it could be used exclusively for cell growth and maintenance. We show that the two modules are functionally independent from each other, allowing efficient lipid accumulation while maintaining active cell growth. Our strategy resulted in 7.2‐ and 4.2‐fold improvements in WE content and productivity, respectively, and the product titer was enhanced by 8.3‐fold over the wild type strain. Notably, during a 24‐h cultivation, a yield of 18% C‐WE/C‐total‐substrates was achieved, being the highest reported for WE biosynthesis. This study provides a simple, yet powerful, means of controlling cellular operations and overcoming some of the fundamental challenges in microbial storage lipid production. [ABSTRACT FROM AUTHOR]

Lignin has potential as a sustainable feedstock for microbial production of industrially relevant molecules. However, the required lignin depolymerization yields a heterogenic mixture of aromatic monomers that are challenging substrates for the microorganisms commonly used in the industry. Here, we investigated the properties of lignin‐related aromatic compounds (LRAs), namely coumarate, ferulate, and caffeate, in the synthesis of biomass and products in an LRA‐utilizing bacterial host Acinetobacter baylyi ADP1. The biosynthesis products, wax esters, and alkanes are relevant compounds for the chemical and fuel industries. Here, wax esters were produced by a native pathway of ADP1, whereas alkanes were produced by a synthetic pathway introduced to the host. Using individual LRAs as substrates, the growth and product formation were monitored with internal biosensors and off‐line analytics. Of the tested LRAs, coumarate was the most propitious in terms of product synthesis. Wax esters were produced from coumarate with yield and titer of 37 mg/gcoumarate and 202 mg/L, whereas alkanes were produced with a yield of 62.3 µg /gcoumarate and titer of 152 µg/L. This study demonstrates the microbial preference for certain LRAs and highlights the potential of A. baylyi ADP1 as a host for LRA upgrading to value‐added products. [ABSTRACT FROM AUTHOR]