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1. Machine learning-guided acyl-ACP reductase engineering for improved in vivo fatty alcohol production

   https://doi.org/10.1038/s41467-021-25831-w  

   Greenhalgh, Jonathan C.; Fahlberg, Sarah A.; Pfleger, Brian F.; Romero, Philip A. (December 2021, Nature Communications)

   null (Ed.)

   Abstract Alcohol-forming fatty acyl reductases (FARs) catalyze the reduction of thioesters to alcohols and are key enzymes for microbial production of fatty alcohols. Many metabolic engineering strategies utilize FARs to produce fatty alcohols from intracellular acyl-CoA and acyl-ACP pools; however, enzyme activity, especially on acyl-ACPs, remains a significant bottleneck to high-flux production. Here, we engineer FARs with enhanced activity on acyl-ACP substrates by implementing a machine learning (ML)-driven approach to iteratively search the protein fitness landscape. Over the course of ten design-test-learn rounds, we engineer enzymes that produce over twofold more fatty alcohols than the starting natural sequences. We characterize the top sequence and show that it has an enhanced catalytic rate on palmitoyl-ACP. Finally, we analyze the sequence-function data to identify features, like the net charge near the substrate-binding site, that correlate with in vivo activity. This work demonstrates the power of ML to navigate the fitness landscape of traditionally difficult-to-engineer proteins. 

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2. Arabidopsis ACYL CARRIER PROTEIN4 and RHOMBOID LIKE10 act independently in chloroplast phosphatidate synthesis

   https://doi.org/10.1093/plphys/kiad483  

   Xu, Yang; Kambhampati, Shrikaar; Morley, Stewart A; Cook, Ron; Froehlich, John; Allen, Doug K; Benning, Christoph (September 2023, Plant Physiology)

   Abstract ACYL CARRIER PROTEIN4 (ACP4) is the most abundant ACP isoform in Arabidopsis (Arabidopsis thaliana) leaves and acts as a scaffold for de novo fatty acid biosynthesis and as a substrate for acyl-ACP-utilizing enzymes. Recently, ACP4 was found to interact with a protein-designated plastid RHOMBOID LIKE10 (RBL10) that affects chloroplast monogalactosyldiacylglycerol (MGDG) biosynthesis, but the cellular function of this interaction remains to be explored. Here, we generated and characterized acp4 rbl10 double mutants to explore whether ACP4 and RBL10 directly interact in influencing chloroplast lipid metabolism. Alterations in the content and molecular species of chloroplast lipids such as MGDG and phosphatidylglycerol were observed in the acp4 and rbl10 mutants, which are likely associated with the changes in the size and profiles of diacylglycerol (DAG), phosphatidic acid (PA), and acyl-ACP precursor pools. ACP4 contributed to the size and profile of the acyl-ACP pool and interacted with acyl-ACP-utilizing enzymes, as expected for its role in fatty acid biosynthesis and chloroplast lipid assembly. RBL10 appeared to be involved in the conversion of PA to DAG precursors for MGDG biosynthesis as evidenced by the increased 34:x PA and decreased 34:x DAG in the rbl10 mutant and the slow turnover of radiolabeled PA in isolated chloroplasts fed with [14C] acetate. Interestingly, the impaired PA turnover in rbl10 was partially reversed in the acp4 rbl10 double mutant. Collectively, this study shows that ACP4 and RBL10 affect chloroplast lipid biosynthesis by modulating substrate precursor pools and appear to act independently. 

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3. Metabolic engineering strategies to produce medium-chain oleochemicals via acyl-ACP:CoA transacylase activity

   https://doi.org/10.1038/s41467-022-29218-3  

   Yan, Qiang; Cordell, William T.; Jindra, Michael A.; Courtney, Dylan K.; Kuckuk, Madeline K.; Chen, Xuanqi; Pfleger, Brian F. (December 2022, Nature Communications)

   Abstract Microbial lipid metabolism is an attractive route for producing oleochemicals. The predominant strategy centers on heterologous thioesterases to synthesize desired chain-length fatty acids. To convert acids to oleochemicals (e.g., fatty alcohols, ketones), the narrowed fatty acid pool needs to be reactivated as coenzyme A thioesters at cost of one ATP per reactivation - an expense that could be saved if the acyl-chain was directly transferred from ACP- to CoA-thioester. Here, we demonstrate such an alternative acyl-transferase strategy by heterologous expression of PhaG, an enzyme first identified inPseudomonads, that transfers 3-hydroxy acyl-chains between acyl-carrier protein and coenzyme A thioester forms for creating polyhydroxyalkanoate monomers. We use it to create a pool of acyl-CoA’s that can be redirected to oleochemical products. Through bioprospecting, mutagenesis, and metabolic engineering, we develop three strains ofEscherichia colicapable of producing over 1 g/L of medium-chain free fatty acids, fatty alcohols, and methyl ketones. 

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4. Exploring the compatibility of phosphopantetheinyl transferases with acyl carrier proteins spanning type II polyketide synthase sequence space

   https://doi.org/10.1093/jimb/kuaf031  

   Bifendeh, Areta_L N; Hsu, Kenneth K; McBride, Christina M; Ferguson, Charlie M; Baumann, Eva R; Capcha-Rodriguez, Diego; Chen, Xinnuo; Chery, Berlensie; Chihade, Margo M; Umpierre, Paola Delgado; et al (September 2025, Journal of Industrial Microbiology and Biotechnology)

   Abstract Phosphopantetheinyl transferases (PPTases) play an essential role in primary and secondary metabolism. These enzymes facilitate the post-translational activation of acyl carrier proteins (ACPs) central to the biosynthesis of fatty acids and polyketides. Modulation of ACP-PPTase interactions is a promising approach to both increase access to desired molecular outputs and disrupt mechanisms associated with disease progression. However, such an approach requires understanding the molecular principles that govern ACP-PPTase interactions across diverse synthases. Through a multi-year, course-based undergraduate research experience (CURE), 17 ACPs representing a range of putative type II polyketide synthases, from actinobacterial and non-actinobacterial phyla, were evaluated as substrates for three PPTases (AcpS, Sfp, and vulPPT). The observed PPTase compatibility, sequence-level analyses, and predictive structural modelling suggest that ACP selectivity is driven by amino acids surrounding the conserved, modified serine on the ACP. We propose that vulPPT and Sfp are driven primarily by hydrophobic contacts, whereas AcpS may favor ACPs which exhibit high net-negative charge density, as well as a broad electronegative surface distribution. Furthermore, we report a plausible, hitherto unreported hydrophobic interaction between vulPPT and a conserved ACP crease, upstream of the invariant serine, which may facilitate docking. This work provides a catalog of compatible and incompatible ACP-PPTase partnerships, highlighting specific regions on the ACP and/or PPTase that show promise for future strategic engineering and inhibitor development efforts. 

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5. Involvement of Arabidopsis Acyl Carrier Protein 1 in PAMP-triggered immunity

   https://doi.org/10.1094/MPMI-02-22-0049-R  

   Zhao, Zhenzhen; Fan, Jiangbo; Yang, Piao; Wang, Zonghua; Stephen, Opiyo; Mackey, David; Ye, Xia (January 2022, Molecular Plant-Microbe Interactions®)

   Plant fatty acids (FAs) and lipids are essential in storing energy and act as structural components for cell membranes and signaling molecules for plant growth and stress responses. Acyl Carrier Proteins (ACPs) are small acidic proteins that covalently bind the fatty acyl intermediates during the elongation of FAs. The Arabidopsis thaliana ACP family has eight members. Through reverse genetic, molecular, and biochemical approaches, we have discovered that ACP1 localizes to the chloroplast and limits the magnitude of pattern-triggered immunity (PTI) against the bacterial pathogen Pseudomonas syringae pathovar tomato (Pto). The mutant acp1 plants have reduced levels of linolenic acid (18:3), which is the primary precursor for the biosynthesis of the phytohormone jasmonic acid (JA), and a corresponding decrease in the abundance of JA. Consistent with the known antagonistic relationship between JA and salicylic acid (SA), acp1 mutant plants also accumulate higher level of SA and display the corresponding shifts in JA- and SA-regulated transcriptional outputs. Moreover, the methyl JA and linolenic acid treatments cause an apparently enhanced decrease of resistance against Pto in acp1 mutants than that in wild-type plants. The ability of ACP1 to prevent this hormone imbalance likely underlies its negative impact on PTI in plant defense. Thus, ACP1 links FA metabolism to stress hormone homeostasis to be negatively involved in PTI in Arabidopsis plant defense. 

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