scholarly journals Machine learning-guided acyl-ACP reductase engineering for improved in vivo fatty alcohol production

2021 ◽  
Author(s):  
Jonathan C Greenhalgh ◽  
Sarah A Fahlberg ◽  
Brian F Pfleger ◽  
Philip A Romero
Keyword(s):  
Machine Learning ◽  
Fatty Acid ◽  
Fatty Alcohol ◽  
Fatty Acid Biosynthesis ◽  
Enzyme Engineering ◽  
Fatty Alcohols ◽  
Energetic Cost ◽  
Acid Biosynthesis ◽  
Alcohol Production

Fatty acyl reductases (FARs) catalyze the reduction of thioesters to alcohols and are key enzymes for the microbial production of fatty alcohols. Many existing metabolic engineering strategies utilize these reductases to produce fatty alcohols from intracellular acyl-CoA pools; however, acting on acyl-ACPs from fatty acid biosynthesis has a lower energetic cost and could enable more efficient production of fatty alcohols. Here we engineer FARs to preferentially act on acyl-ACP substrates and produce fatty alcohols directly from the fatty acid biosynthesis pathway. We implemented a machine learning-driven approach to iteratively search the protein fitness landscape for enzymes that produce high titers of fatty alcohols in vivo. After ten design-test-learn rounds, our approach converged on engineered enzymes that produce over twofold more fatty alcohols than the starting natural sequences. We further characterized the top identified sequence and found its improved alcohol production was a result of an enhanced catalytic rate on acyl-ACP substrates, rather than enzyme expression or KM effects. Finally, we analyzed the sequence-function data generated during the enzyme engineering to identify sequence and structure features that influence fatty alcohol production. We found an enzyme's net charge near the substrate-binding site was strongly correlated with in vivo activity on acyl-ACP substrates. These findings suggest future rational design strategies to engineer highly active enzymes for fatty alcohol production.

scholarly journals Evolution and Functional Characteristics of the Novel Elovl8 That Play Pivotal Roles in Fatty Acid Biosynthesis

Genes ◽  
2021 ◽  
Vol 12 (8) ◽  
pp. 1287
Author(s):  
Shouxiang Sun ◽  
Yumei Wang ◽  
Pei-Tian Goh ◽  
Mónica Lopes-Marques ◽  
L. Filipe C. Castro ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Biosynthesis ◽  
The Novel ◽  
Acid Biosynthesis ◽  
Long Chain Fatty Acid ◽  
Rate Limiting Step ◽  
Gene Expression Quantification ◽  
Rate Limiting ◽  
Zebrafish Liver

Elongation of very long-chain fatty acid (Elovl) proteins are key enzymes that catalyze the rate-limiting step in the fatty acid elongation pathway. The most recently discovered member of the Elovl family, Elovl8, has been proposed to be a fish-specific elongase with two gene paralogs described in teleosts. However, the biological functions of Elovl8 are still to be elucidated. In this study, we showed that in contrast to previous findings, elovl8 is not unique to teleosts, but displays a rather unique and ample phylogenetic distribution. For functional determination, we generated elovl8a (elovl8a−/−) and elovl8b (elovl8b−/−) zebrafish using CRISPR/Cas9 technology. Fatty acid composition in vivo and zebrafish liver cell experiments suggest that the substrate preference of Elovl8 overlapped with other existing Elovl enzymes. Zebrafish Elovl8a could elongate the polyunsaturated fatty acids (PUFAs) C18:2n-6 and C18:3n-3 to C20:2n-6 and C20:3n-3, respectively. Along with PUFA, zebrafish Elovl8b also showed the capacity to elongate C18:0 and C20:1. Gene expression quantification suggests that Elovl8a and Elovl8b may play a potentially important role in fatty acid biosynthesis. Overall, our results provide novel insights into the function of Elovl8a and Elovl8b, representing additional fatty acid elongases not previously described in chordates.

scholarly journals Biosynthesis of Fatty Alcohols in Engineered Microbial Cell Factories: Advances and Limitations

2020 ◽  
Vol 8 ◽  
Author(s):  
Anagha Krishnan ◽  
Bonnie A. McNeil ◽  
David T. Stuart
Keyword(s):  
Fatty Acid ◽  
Fatty Alcohol ◽  
Industrial Applications ◽  
Microbial Cell ◽  
Fatty Alcohols ◽  
Scale Production ◽  
Endogenous Fatty Acid ◽  
Alcohol Production ◽  
Microbial Cell Factories ◽  
Cell Factories

Concerns about climate change and environmental destruction have led to interest in technologies that can replace fossil fuels and petrochemicals with compounds derived from sustainable sources that have lower environmental impact. Fatty alcohols produced by chemical synthesis from ethylene or by chemical conversion of plant oils have a large range of industrial applications. These chemicals can be synthesized through biological routes but their free forms are produced in trace amounts naturally. This review focuses on how genetic engineering of endogenous fatty acid metabolism and heterologous expression of fatty alcohol producing enzymes have come together resulting in the current state of the field for production of fatty alcohols by microbial cell factories. We provide an overview of endogenous fatty acid synthesis, enzymatic methods of conversion to fatty alcohols and review the research to date on microbial fatty alcohol production. The primary focus is on work performed in the model microorganisms, Escherichia coli and Saccharomyces cerevisiae but advances made with cyanobacteria and oleaginous yeasts are also considered. The limitations to production of fatty alcohols by microbial cell factories are detailed along with consideration to potential research directions that may aid in achieving viable commercial scale production of fatty alcohols from renewable feedstock.

scholarly journals Proteomic Signature of Fatty Acid Biosynthesis Inhibition Available for In Vivo Mechanism-of-Action Studies

2011 ◽  
Vol 55 (6) ◽  
pp. 2590-2596 ◽  
Author(s):  
Michaela Wenzel ◽  
Malay Patra ◽  
Dirk Albrecht ◽  
David Y.-K. Chen ◽  
K. C. Nicolaou ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Biosynthesis ◽  
Structure Activity Relationship ◽  
Mechanisms Of Action ◽  
Activity Relationship ◽  
Antibacterial Drug ◽  
Acid Biosynthesis ◽  
Content Type ◽  
Structure Activity

ABSTRACTFatty acid biosynthesis is a promising novel antibiotic target. Two inhibitors of fatty acid biosynthesis, platencin and platensimycin, were recently discovered and their molecular targets identified. Numerous structure-activity relationship studies for both platencin and platensimycin are currently being undertaken. We established a proteomic signature for fatty acid biosynthesis inhibition inBacillus subtilisusing platencin, platensimycin, cerulenin, and triclosan. The induced proteins, FabHA, FabHB, FabF, FabI, PlsX, and PanB, are enzymes involved in fatty acid biosynthesis and thus linked directly to the target pathway. The proteomic signature can now be used to assess thein vivomechanisms of action of compounds derived from structure-activity relationship programs, as demonstrated for the platensimycin-inspired chromium bioorganometallic PM47. It will further serve as a reference signature for structurally novel natural and synthetic antimicrobial compounds with unknown mechanisms of action. In summary, we described a proteomic signature inB. subtilisconsisting of six upregulated proteins that is diagnostic of fatty acid biosynthesis inhibition and thus can be applied to advance antibacterial drug discovery programs.

scholarly journals In vivo and in vitro effects of thiolactomycin on fatty acid biosynthesis in Streptomyces collinus.

1997 ◽  
Vol 179 (12) ◽  
pp. 3884-3891 ◽  
Author(s):  
K K Wallace ◽  
S Lobo ◽  
L Han ◽  
H A McArthur ◽  
K A Reynolds
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Biosynthesis ◽  
Acid Biosynthesis ◽  
Streptomyces Collinus ◽  
In Vitro Effects

scholarly journals Evaluating the effects of synthetic POM cycles and NAD kinase expression on fatty alcohol production in Saccharomyces cerevisiae

2020 ◽  
Author(s):  
Bonnie A McNeil ◽  
Charfeddine Khalifa ◽  
Anagha Krishnan ◽  
David T Stuart
Keyword(s):  
Fatty Alcohol ◽  
Carbon Flux ◽  
Metabolic Flux ◽  
Fatty Acid Biosynthesis ◽  
Nad Kinase ◽  
Acid Biosynthesis ◽  
Alcohol Production ◽  
Fatty Acid Biosynthesis Pathway ◽  
Kinase Expression ◽  
Background Expression

Abstract Background: NADPH-dependent enzymes play important roles in many anabolic reactions and the availability of redox cofactors can influence metabolic flux ultimately influencing titers of bioproducts produced by engineered microbial cells. This may be especially true of oleochemical production when carbon flux through the highly NADPH-dependent fatty acid biosynthesis pathway is increased. While pathway specific approaches are often applied to counter redox imbalance, a study evaluating generalized approaches to improved NADPH availability is lacking in Saccharomyces cerevisiae . Results: Here, we have created four unique synthetic Pyruvate-Oxaloacetate-Malate “POM” cycles consisting of either of the endogenous isoforms of pyruvate carboxylase ( PYC1 or PYC2 ), a modified version of malate dehydrogenase ( ‘MDH1 or ‘MDH2 ), and a truncated cytosolic form of the endogenous malic enzyme ( sMAE1 ). Only the POM cycle that combined expression of PYC1 , ‘MDH2 , and sMAE1 increased the titer of fatty alcohols produced; however, it did so in two unique fatty alcohol producing strains. In a FAS1 overexpression background, expression of this synthetic POM cycle increased fatty alcohol titers by 40% from 49.0 ± 2.2 mg/L to 68.6 ± 3.3 mg/L and showed similar results in a zwf1 deletion strain. The effect of overexpression of the endogenous NAD+ kinases UTR1 , YEF1 , and a cytosolic version of POS5 were also tested. We found that expression of POS5c resulted in an ~35% increase in fatty alcohol titer, while the overexpression of the UTR1 or YEF1 did not significantly influence titers. In these minimally engineered cells, combined overexpression of PYC1 , ‘ MDH2 , sMAE1 and POS5c did not further increase titers Conclusions: Overexpression of PYC1 in conjunction with ‘MDH2 and sMAE1 results in a synthetic POM cycle which can be utilized to improve fatty alcohol production in engineered strains of S. cerevisiae . Additionally, overexpression of a truncated version of POS5 ( POS5c ) results in similar increases in fatty alcohol production. These findings may serve to provide a generalized mechanism to increase NADPH production in engineered cells, resulting in increased bioproduct titers.

scholarly journals Slow Onset Inhibitors of Bacterial Fatty Acid Biosynthesis: Residence Time, In Vivo Activity and In Vivo Imaging

2010 ◽  
Vol 24 (S1) ◽  
Author(s):  
Peter J Tonge ◽  
Christopher Ende ◽  
Suan Knudson ◽  
Sylvia Luckner ◽  
B Gopal Reddy ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Residence Time ◽  
In Vivo Imaging ◽  
Fatty Acid Biosynthesis ◽  
Acid Biosynthesis ◽  
Bacterial Fatty Acid ◽  
Slow Onset

scholarly journals In Vivo Roles of Fatty Acid Biosynthesis Enzymes in Biosynthesis of Biotin and α-Lipoic Acid in Corynebacterium glutamicum

2017 ◽  
Vol 83 (19) ◽  
Author(s):  
Masato Ikeda ◽  
Takashi Nagashima ◽  
Eri Nakamura ◽  
Ryosuke Kato ◽  
Masakazu Ohshita ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Lipoic Acid ◽  
Fatty Acid Synthase ◽  
Fatty Acid Biosynthesis ◽  
Physiological Role ◽  
Type I ◽  
Acid Biosynthesis ◽  
Content Type ◽  
Fas Ii

ABSTRACT For fatty acid biosynthesis, Corynebacterium glutamicum uses two type I fatty acid synthases (FAS-I), FasA and FasB, in addition to acetyl-coenzyme A (CoA) carboxylase (ACC) consisting of AccBC, AccD1, and AccE. The in vivo roles of the enzymes in supplying precursors for biotin and α-lipoic acid remain unclear. Here, we report genetic evidence demonstrating that the biosynthesis of these cofactors is linked to fatty acid biosynthesis through the FAS-I pathway. For this study, we used wild-type C. glutamicum and its derived biotin vitamer producer BFI-5, which was engineered to express Escherichia coli bioBF and Bacillus subtilis bioI. Disruption of either fasA or fasB in strain BFI-5 led to decreased production of biotin vitamers, whereas its amplification contributed to increased production, with a larger impact of fasA in both cases. Double disruptions of fasA and fasB resulted in no biotin vitamer production. The acc genes showed a positive effect on production when amplified simultaneously. Augmented fatty acid biosynthesis was also reflected in pimelic acid production when carbon flow was blocked at the BioF reaction. These results indicate that carbon flow down the FAS-I pathway is destined for channeling into the biotin biosynthesis pathway, and that FasA in particular has a significant impact on precursor supply. In contrast, fasB disruption resulted in auxotrophy for lipoic acid or its precursor octanoic acid in both wild-type and BFI-5 strains. The phenotypes were fully complemented by plasmid-mediated expression of fasB but not fasA. These results reveal that FasB plays a specific physiological role in lipoic acid biosynthesis in C. glutamicum. IMPORTANCE For the de novo biosynthesis of fatty acids, C. glutamicum exceptionally uses a eukaryotic multifunctional type I fatty acid synthase (FAS-I) system comprising FasA and FasB, in contrast to most bacteria, such as E. coli and B. subtilis, which use an individual nonaggregating type II fatty acid synthase (FAS-II) system. In this study, we reported genetic evidence demonstrating that the FAS-I system is the source of the biotin precursor in vivo in the engineered biotin-prototrophic C. glutamicum strain. This study also uncovered the important physiological role of FasB in lipoic acid biosynthesis. Here, we present an FAS-I enzyme that functions in supplying the lipoic acid precursor, although its biosynthesis has been believed to exclusively depend on FAS-II in organisms. The findings obtained here provide new insights into the metabolic engineering of this industrially important microorganism to produce these compounds effectively.

scholarly journals In vivo analysis of straight-chain and branched-chain fatty acid biosynthesis in three actinomycetes

1995 ◽  
Vol 131 (2) ◽  
pp. 227-234 ◽  
Author(s):  
Kimberlee K. Wallace ◽  
Bitao Zhao ◽  
Hamish A.I. McArthur ◽  
Kevin A. Reynolds
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Biosynthesis ◽  
Chain Fatty Acid ◽  
Acid Biosynthesis ◽  
Straight Chain ◽  
In Vivo Analysis ◽  
Branched Chain
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